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Bombyx mori is an insect species of great economic importance, and its silk gland is a vital organ for the synthesis and secretion of silk protein. However, long-term artificial domestication of B. mori has resulted in high sensitivity to chemical toxins, especially insecticides. Cyantraniliprole (Cya), a second-generation ryanodine receptor modulator insecticide, is widely utilized in agriculture for pest control. In this study, the impact of Cya toxicity on the development of silk glands in the 5th instar larvae of B. mori was assessed using Cya LC 5 , LC 10 and LC 20 , as well as a starvation treatment group for comparison. Short-term exposure (24 h) to different concentrations of Cya resulted in delayed development of silk glands in B. mori. Meanwhile, the body weight, silk gland weight, silk gland index and cocoon quality were significantly reduced in a concentration-dependent manner, except for the Cya LC 5 treatment. Histopathological and ultrastructural analysis revealed that Cya LC 10 induced disruption of the nuclear membrane and endoplasmic reticulum in the posterior silk gland (PSG) cells, leading to the formation of intracellular vacuoles. Transcriptome sequencing of PSGs identified 2152 genes that were differentially expressed after exposure to Cya LC 10 , with 1153 down-regulated genes and 999 up-regulated genes. All differentially expressed genes were subjected to functional annotation using gene ontology and Kyoto encyclopedia of genes and genomes database, and it was found that protein synthesis-related pathways were significantly enriched, with the majority of genes being down-regulated. Furthermore, the transcription levels of genes involved in "protein processing in endoplasmic reticulum", "protein export", "proteasome" and "DNA replication" were quantified using qRT-PCR. Our findings suggested that short-term exposure to Cya LC 10 resulted in disruption of DNA replication, as well as protein transport, processing and hydrolysis in the PSG cells of B. mori. The results of this study provide a theoretical foundation for the safe utilization of Cya in sericulture production., 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. Published by Elsevier Inc.)

Our previous research reported the influence of 50 μM selenium (Se) on the cytosolization (endocytosis) pathway, which in turn stimulates the growth and development of Bombyx mori. Lately, dynamin is recognized as one of the key proteins in endocytosis. To explore the underlying mechanisms of Se impact, the dynamin gene was knocked down by injecting siRNAs (Dynamin-1, Dynamin-2, and Dynamin-3). This was followed by an analysis of the target gene and levels of silk protein genes, as well as growth and developmental indices, Se-enrichment capacity, degree of oxidative damage, and antioxidant capacity of B. mori. Our findings showed a considerable decrease in the relative expression of the dynamin gene in all tissues 24 h after the interference and a dramatic decrease in the silkworm body after 48 h. RNAi dynamin gene decreased the silkworm body weight, cocoon shell weight, and the ratio of cocoon. In the meantime, malondialdehyde level increased and glutathione level and superoxide dismutase/catalase activities decreased. 50 μM Se markedly ameliorated these growth and physiological deficits as well as decreases in dynamin gene expression. On the other hand, there were no significant effects on fertility (including produced eggs and laid eggs) between the interference and Se treatments. Additionally, the Se content in the B. mori increased after the dynamin gene interference. The dynamin gene was highly expressed in the silk gland and declined significantly after interference. Among the three siRNAs (Dynamin-1, Dynamin-2, and Dynamin-3), the dynamin-2 displayed the highest interference effects to target gene expression. Our results demonstrated that 50 μM Se was effective to prevent any adverse effects caused by dynamin knockdown in silkworms. This provides practical implications for B. mori breeding industry., (© 2024 Wiley Periodicals LLC.)

Background: Bombyx mori nuclear polyhedrosis virus (BmNPV), as a typical baculovirus, is the primary pathogen that infects the silkworm B. mori, a lepidopteran species. Owing to the high biological safety of BmNPV in infecting insects, it is commonly utilized as a biological insecticide for pest control. Apoptosis is important in the interaction between the host and pathogenic microorganisms. MicroRNAs (miRNAs) influence immune responses and promote stability of the immune system via apoptosis. Therefore, the study of apoptosis-related miRNA in silkworms during virus infection can not only provide support for standardizing the prevention and control of diseases and insect pests, but also reduce the economic losses to sericulture caused by the misuse of biological pesticides., Results: Through transcriptome sequencing, we identified a miRNA, miR-31-5p, and demonstrated that it can inhibit apoptosis in silkworm cells and promote the proliferation of BmNPV in BmE-SWU1 cells. We identified a target gene of miR-31-5p, B. mori cytochrome P450 9e2 (BmCYP9e2), and demonstrated that it can promote apoptosis in silkworm cells and inhibit the proliferation of BmNPV. Moreover, we constructed transgenic silkworm strains with miR-31-5p knockout and confirmed that they can inhibit the proliferation of BmNPV., Conclusion: These data indicate that miR-31-5p may exert functions of inhibiting apoptosis and promoting virus proliferation by regulating BmCYP9e2. The findings demonstrate how miRNAs influence host cell apoptosis and how they are involved in the host immune system response to viruses, providing important insights into the applications of biological insecticides for pest control. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Most insects enter diapause, a state of physiological dormancy crucial for enduring harsh seasons, with photoperiod serving as the primary cue for its induction, ensuring proper seasonal timing of the process. Although the involvement of the circadian clock in the photoperiodic time measurement has been demonstrated through knockdown or knockout of clock genes, the involvement of clock gene cryptochrome 1 (cry1), which functions as a photoreceptor implicated in photoentrainment of the circadian clock across various insect species, remains unclear. In bivoltine strains of the silkworm, Bombyx mori, embryonic diapause is maternally controlled and affected by environmental conditions experienced by mother moths during embryonic and larval stages. Previous research highlighted the role of core clock genes, including period (per), timeless (tim), Clock (Clk) and cycle (cyc), in photoperiodic diapause induction in B. mori. In this study, we focused on the involvement of cry1 gene in B. mori photoperiodism. Phylogenetic analysis and conserved domain identification confirmed the presence of both Drosophila-type cry (cry1) and mammalian-type cry (cry2) genes in the B. mori genome, akin to other lepidopterans. Temporal expression analysis revealed higher cry1 gene expression during the photophase and lower expression during the scotophase, with knockouts of core clock genes (per, tim, Clk and cyc) disrupting this temporal expression pattern. Using CRISPR/Cas9-mediated genome editing, we established a cry1 knockout strain in p50T, a bivoltine strain exhibiting clear photoperiodism during both embryonic and larval stages. Although the wild-type strain displayed circadian rhythm in eclosion under continuous darkness, the cry1 knockout strain exhibited arrhythmic eclosion, implicating B. mori cry1 in the circadian clock feedback loop governing behavior rhythms. Females of the cry1 knockout strain failed to control photoperiodic diapause induction during both embryonic and larval stages, mirroring the diapause phenotype of the wild-type individuals reared under constant darkness, indicating that B. mori CRY1 contributes to photoperiodic time measurement as a photoreceptor. Furthermore, photoperiodic diapause induction during the larval stage was abolished in a cry1/tim double-knockout strain, suggesting that photic information received by CRY1 is relayed to the circadian clock. Overall, this study represents the first evidence of cry1 involvement in insect photoperiodism, specifically in diapause induction., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The prothoracic gland (PG) is the source of ecdysteoids in larval insects. Although numerous studies have been conducted on signaling networks involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in PGs, less is known about regulation of metabolism in PGs. In the present study, we investigated correlations between expressions of sugar transporter (St)/trehalase (Treh) genes and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that in vitro PTTH treatment stimulated expression of the St1 gene, but not other transporter genes. Expression of the Treh1 gene was also stimulated by PTTH treatment. An immunoblotting analysis showed that St1 protein levels in Bombyx PGs increased during the later stage of the last larval instar and were not affect by PTTH treatment. PTTH treatment enhanced Treh enzyme activity in a time-dependent manner. Blocking either extracellular signal-regulated kinase (ERK) signaling with U0126 or phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 decreased PTTH-stimulated Treh enzyme activity, indicating a link from the ERK and PI3K signaling pathways to Treh activity. Treatment with the Treh inhibitor, validamycin A, blocked PTTH-stimulated Treh enzyme activity and partially inhibited PTTH-stimulated ecdysteroidogenesis. Treatment with either a sugar transport inhibitor (cytochalasin B) or a specific glycolysis inhibitor (2-deoxy-D-glucose, 2-DG) partially inhibited PTTH-stimulated ecdysteroidogenesis. Taken together, these results indicate that increased expressions of St1/Treh1 and Treh activity, which lie downstream of PTTH signaling, are involved in PTTH stimulation in B. mori PGs., 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 Ltd. All rights reserved.)

Insect developmental transitions are precisely coordinated by ecdysone and juvenile hormone (JH). We previously revealed that accumulated H3K27 trimethylation (H3K27me3) at the locus encoding JH signal transducer Hairy is involved in the larval-pupal transition in insects, but the underlying mechanism remains to be fully defined. Here, we show in Drosophila and Bombyx that Rpd3-mediated H3K27 deacetylation in the prothoracic gland during the last larval instar promotes ecdysone biosynthesis and the larval-pupal transition by enabling H3K27me3 accumulation at the Hairy locus to induce its transcriptional repression. Importantly, we find that the homeodomain transcription factor Schlank acts to switch active H3K27 acetylation (H3K27ac) to repressive H3K27me3 at the Hairy locus by directly binding to the Hairy promoter and then recruiting the histone deacetylase Rpd3 and the histone methyltransferase PRC2 component Su(z)12 through physical interactions. Moreover, Schlank inhibits Hairy transcription to facilitate the larval-pupal transition, and the Schlank signaling cascade is suppressed by JH but regulated in a positive feedback manner by ecdysone. Together, our data uncover that Schlank mediates epigenetic reprogramming of H3K27 modifications in hormone actions during insect developmental transition., Competing Interests: Competing interests statement:The authors declare no competing interest.

The egg parasitoid Mesocomys trabalae Yao, Yang, and Zhao is used as a biocontrol agent against the emerging defoliator pest Caligula japonica Moore in East Asia. It has been proven that the eggs of Antheraea pernyi Guérin-Méneville can be used as a factitious host for the mass production of M. trabalae. We examined the parasitic behavior and morphological characteristics of the developmental stages of M. trabalae reared on A. pernyi eggs. The parasitic behavior of M. trabalae encompasses 10 steps, involving searching, antennation, locating the oviposition site, drilling, probing, detecting, oviposition, host feeding, grooming, and resting, with the oviposition step further divided into 3 stages. We determined that the parasitoid released an egg during the second stage of the oviposition step, while her body remained in a relatively static state. Among all the steps in parasitic behavior, probing occupied the longest time, accounting for 26.33% of the entire parasitism process. It was followed by oviposition (15.88%), drilling (15.10%), antennation (13.09%), detecting (10.79%), host feeding (10.02%), and the remaining steps, each occupying less than 5.00% of the total time in steps. The pre-emergence of adult M. trabalae comprised of 4 stages: egg (0-1 day), larva (2-6 days), prepupa (7-11 days), pupa (12-20 days), followed by the development into an adult, and it usually took 20-22 days to develop from an egg into an adult at 25°C. This study advances our understanding of the biology of Mesocomys parasitoids and their mass-rearing for use in augmentation programs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The ovary generally undergoes tissue remodeling during larval to pupal transition, which includes membrane degeneration and ovariole growth. At the same time, the hormones produced by insects significantly change during metamorphosis. However, the regulatory mechanism for ovarian development and hormones is not fully understood in insects. Herein, we found that matrix metalloproteinase 2 (MMP2) was highly expressed in the ovarian capsules and ovarioles, and the development was abnormal after knocking out MMP2 in Bombyx mori. The process of abnormal degradation of collagen I due to MMP2 deletion, which resulted in abnormal development of ovarioles and eggs, was analyzed in detail. The proteomics of ovaries in the MMP2-knock out and wild type strains showed a critically significant difference in the expression of a protein, insulin-like peptide (ILP). Additional analysis revealed significant alteration of ILP during ovarian development, and abnormal expression of ILP significantly affected ovarian development in vivo and MMP2 expression in vitro and in vivo. These results showed that MMP2 regulation of ovarian tissue remodeling is closely related to ILP expression. Our study provides new insights into the regulatory mechanism of MMP2 and ovarian development in B. mori., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

JH and ecdysone signaling regulate insect metamorphosis through the master transcription factors, Krüppel homolog 1 (kr-h1), Broad-Complex (BR-C), and E93. Ecdysone signaling activates successively expressed ecdysone responsive transcription factors (ERTFs), and the interaction between ERTFs determines the expression profiles of ERTFs themselves. Through the construction of expressed sequence tag (EST) database of Bombyx mori from many tissues, the existence of a large number of cuticular protein (CP) genes was identified in wing disc cDNA library of the 3 days after the start of wandering (W3). From the genomic analysis, 12 types of CP clusters of CP genes were identified. DNA sequences of CP genes revealed the duplication of CP genes, which suggests to reflect the insect evolution. These CP genes responded to ecdysone and ecdysone pulse; therefore, CP genes were applied for the analysis of transcriptional regulation by ERTF. The binding sites of ERTF have been reported to exist upstream of CP genes in several insects, and the activation of CP genes occurred by the binding of ERTFs. Through the analysis, the following were speculated; the successive appearance of ERTFs and the activation of target genes resulted in the successively produced CPs and cuticular layer. The sequence of the ERTF and CP gene expression was the same at larval to pupal and pupal to adult transformation. The involvement of several ERTFs in one CP gene expression was also clarified; BmorCPG12 belongs to group showing expression peak at W3 and was regulated by two ERTFs; BHR3 and ßFTZ-F1, BmorCPH2 belongs to group showing expression peak at P0 and was regulated by two ERTFs; ßFTZ-F1 and E74A. The involvement of BHR39 as a negative regulator of CP gene expression was found. Larval, pupal, and adult cuticular layers were supposed to be constructed by the combination of different and similar types of CPs, through the expressed timing of CP genes., (© 2024 Wiley Periodicals LLC.)

The diversity of cell types in the brain and how these change during different developmental stages, remains largely unknown. The life cycle of insects is short and goes through 4 distinct stages including embryonic, larval, pupal, and adult stages. During postembryonic life, the larval brain transforms into a mature adult version after metamorphosis. The silkworm, Bombyx mori, is a lepidopteran model insect. Here, we characterized the brain cell repertoire of larval and adult B. mori by obtaining 50 708 single-cell transcriptomes. Seventeen and 12 cell clusters from larval and adult brains were assigned based on marker genes, respectively. Identified cell types include Kenyon cells, optic lobe cells, monoaminergic neurons, surface glia, and astrocyte glia. We further assessed the cell type compositions of larval and adult brains. We found that the transition from larva to adult resulted in great expansion of glial cells. The glial cell accounted for 49.8% of adult midbrain cells. Compared to flies and ants, the mushroom body kenyon cell is insufficient in B. mori, which accounts for 5.4% and 3.6% in larval and adult brains, respectively. Analysis of neuropeptide expression showed that the abundance and specificity of expression varied among individual neuropeptides. Intriguingly, we found that ion transport peptide was specifically expressed in glial cells of larval and adult brains. The cell atlas dataset provides an important resource to explore cell diversity, neural circuits and genetic profiles., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

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.)

MicroRNAs play critical roles in multiple developmental processes in insects. Our previous study showed that CRISPR/Cas9-mediated knock down of the microRNA let-7 in silkworms increased the size of larvae and silk glands, thereby improving the silk production capacity. In this study, we elucidate the molecular mechanism underlying of let-7 regulates growth. Identification of differentially expressed genes in response to let-7 knock down revealed enrichment of pathways associated with cell proliferation and DNA replication. let-7 dysregulation affected the cell cycle and proliferation of the Bombyx mori cell line BmN. Dual-luciferase and target site mutation assays showed that BmCDK1 is a direct target gene of let-7, with only 1 binding site on its 3'-untranslated region. RNA interference of BmCDK1 inhibited cell proliferation, but this effect was counteracted by co-transfection with let-7 antagomir. Moreover, let-7 knock down induced BmCDK1 expression and promoted cell proliferation in multiple tissues, and further induced endomitosis in the silk gland in vivo. Knock down of BmCDK1 resulted in abnormal formation of a new epidermis, and larval development was arrested at the 2nd or 3rd molt stage. Taken together, our results demonstrated that BmCDK1 is a novel target of let-7 in cell fate determination, possessing potential for improving silk yield in silkworm., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

Silkworm fibroins are natural proteinaceous macromolecules and provide core mechanical properties to silk fibers. The synthesis process of fibroins is posterior silk gland (PSG)-exclusive and appears active at the feeding stage and inactive at the molting stage. However, the molecular mechanisms controlling it remain elusive. Here, the silk gland's physiological and nuclear proteomic features were used to characterize changes in its structure and development from molting to feeding stages. The temporal expression profile and immunofluorescence analyses revealed a synchronous transcriptional on-off mode of fibroin genes. Next, the comparative nuclear proteome of the PSG during the last molting-feeding transition identified 798 differentially abundant proteins (DAPs), including 42 transcription factors and 15 epigenetic factors. Protein-protein interaction network analysis showed a "CTCF-FOX-HOX-SOX" association with activated expressions at the molting stage, suggesting a relatively complex and multifactorial regulation of the PSG at the molting stage. In addition, FAIRE-seq verification indicated "closed" and "open" conformations of fibroin gene promoters at the molting and feeding stages, respectively. Such proteome combined with chromatin accessibility analysis revealed the detailed signature of protein factors involved in the temporal regulation of fibroin synthesis and provided insights into silk gland development as well as silk production in 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 B.V. All rights reserved.)

High temperature stress has long-term negative effects on the growth and development of silkworm (Bombyx mori). Different silkworm varieties show the different tolerance to high temperature. The induction of autophagy is linked to increased thermotolerance in diverse ectothermic organisms. However, the function of autophagy in the thermotolerant and thermosensitive silkworm strains under high-temperature conditions remains unclear. The thermotolerant Liangguang NO.2 and thermosensitive Jingsong × Haoyue strains were used to explore the role of autophagy in thermotolerance. Here, we first found that the larval body weight gain was increased in the thermosensitive Jingsong × Haoyue strain, but there was no difference in the thermotolerant Liangguang NO.2 strain under high temperature conditions. High temperature stress had a negative influence on the cocoon performance in both the Liangguang NO.2 and Jingsong × Haoyue strains. Additionally, the autophagy-related gene Atg5 mRNA expression in the Liangguang NO.2 strain was upregulated by high temperature, while the expression of Atg12 mRNA was reduced in the Jingsong × Haoyue strain. Titers of 20-Hydroxyecdysone and the ultraspiracle 1 mRNA expression in the Liangguang NO.2 strain were upregulated by high temperature, which might be associated with the induction of autophagy. These results demonstrate the potentially regulatory mechanism of autophagy in silkworms' tolerance to high temperature, providing a theoretical basis for exploring the physiological mechanism of thermotolerance in insects., (© 2024 Wiley Periodicals LLC.)

Ubiquitin-fold modifier 1 (UFM1) is attached to protein substrates through the sequential activity of an E1 (UBA5)-E2 (UFC1)-E3 (UFL1) cascade. UFL1 is the E3 ligase for UFMylation in vertebrates. However, there have been no studies on UFL1 in silkworm to date. In this study, we identified a UFL1 ortholog in Bombyx mori genome. Spatio-temporal expression profiles showed that BmUFL1 expression was high in the midgut, epidermis, and testis and in the pupa-adult stage. BmUFL1 knockdown inhibited B. mori nucleopolyhedrovirus (BmNPV) proliferation, while BmUFL1 overexpression promoted BmNPV proliferation. Mechanically, protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling and cell apoptosis are involved in BmUFL1-regulated BmNPV proliferation. Overall, these results suggest that BmUFL1 facilitates BmNPV proliferation in silkworm., (© 2024 The Author(s). Archives of Insect Biochemistry and Physiology published by Wiley Periodicals LLC.)

Carboxypeptidase A has been found across various animal species, yet its activation mechanism during the insect molting process remains elusive. Our study specifically delved into the activation mechanism of carboxypeptidase A (Bm-CPA), identified in Bombyx mori's molting fluid during metamorphosis. Initially, western blotting identified two forms of Bm-CPA, 65 kDa and 54 kDa, in the epidermis of silkworms during the molting stage. Expressing the complete Bm-CPA sequence in Pichia pastoris allowed the identification, via mass spectrometry analysis, of a 75-amino-acid propeptide for the initial hydrolysis process. Subsequently, a 35 kDa form of Bm-CPA emerged in the molting fluid, confirmed as the active form through in vitro assays, demonstrating potent carboxypeptidase A activity and faint carboxypeptidase B activity. Four potential activation sites (including Lys158/Arg159 and Arg177/Arg178) were identified through mass spectrometry and amino acid mutation analysis. RNAi of Bm-CPA indicates its critical role in molting. Finally, the carboxypeptidase inhibitor (Bm-CPI) from silkworm molting fluid was expressed to explore its role in regulating Bm-CPA activity, demonstrating a direct interaction with the 35 kDa Bm-CPA. Our research implies Bm-CPA's potential involvement in the silkworm molting process, suggesting diverse regulatory roles. These findings highlight intricate protein regulation patterns during insect metamorphosis and development., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The cotton bollworm, Helicoverpa armigera, is a significant global agricultural pest, particularly detrimental during its larval feeding period. Insects' odorant receptors (ORs) are crucial for their crop-feeding activities, yet a comprehensive analysis of H. armigera ORs has been lacking, and the influence of hormones on ORs remain understudied. Herein, we conducted a genome-wide study and identified 81 ORs, categorized into 15 distinct groups. Analyses of protein motifs and gene structures revealed both conservation within groups and divergence among them. Comparative gene duplication analysis between H. armigera and Bombyx mori highlighted different duplication patterns. We further investigated subcellular localization and protein interactions within the odorant receptor family, providing valuable insights for future functional and interaction studies of ORs. Specifically, we identified that OR48 and OR75 were abundantly expressed during molting/metamorphosis and feeding stages, respectively. We demonstrated that 20E induced the upregulation of OR48 via EcR, while insulin upregulated OR75 expression through InR. Moreover, 20E induced the translocation of OR48 to the cell membrane, mediating its effects. Functional studies involving the knockdown of OR48 and OR75 revealed their roles in metamorphosis development, with OR48 knockdown resulting in delayed pupation and OR75 knockdown leading to premature pupation. OR48 can promote autophagy and apoptosis in fat body, while OR75 can significantly inhibit apoptosis and autophagy. These findings significantly contribute to our understanding of OR function in H. armigera and shed light on potential avenues for pest control strategies., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The silkworm (Bombyx mori) is a model organism for lepidopteran insects. It is an oligophagous insect that primarily feeds on mulberry leaves and has industrial use for the production of raw silk. The development of artificial diets has provided an alternative nutrient source for silkworms; however, one significant issue is that the production of cocoons is lower in silkworms reared on artificial diets compared with those reared on mulberry leaves. The differences in the silk gland in the late-stage fifth instar silkworm larvae, when silk synthesis is most active, between those raised on artificial diets and mulberry leaves, are unknown. In this study, we identified differences in the transcriptomes of the middle and posterior silk glands of fifth instar day five silkworm larvae reared on artificial diets compared with those reared on mulberry leaves using three strains: Daizo, Nichi01, and J137 × C146. We found that the silk-related genes fibrohexamerin (fhx), fibroin-light-chain (fibL), and fibroin-heavy-chain (fibH) in the middle silk gland, and ser1 in the posterior silk gland, were differentially expressed in a strain-dependent manner. In silkworms reared on artificial diets, fhx, fibL, and fibH in the middle silk gland were upregulated in Nichi01 and downregulated in J137 × C146, whereas ser1 in the posterior silk gland was upregulated in J137 × C146 compared with silkworms reared on mulberry leaves. Our results demonstrate that the diet and strain of silkworm larvae affect the expression of genes related to silk production in their silk glands during the late fifth instar stage., 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 Ltd. All rights reserved.)

Time-course transcriptome expression data were constructed for four parts of the silk gland (anterior, middle, and posterior parts of the middle silk gland, along with the posterior silk gland) in the domestic silkworm, Bombyx mori, from days 0 to 7 of the last-instar larvae. For sample preparation, silk glands were extracted from one female and one male larva every 24 hours accurately after the fourth ecdysis. The reliability of these transcriptome data was confirmed by comparing the transcripts per million (TPM) values of the silk gene and quantitative reverse transcription PCR results. Hierarchical cluster analysis results supported the reliability of transcriptome data. These data are likely to contribute to the progress in molecular biology and genetic research using B. mori, such as elucidating the mechanism underlying the massive production of silk proteins, conducting entomological research using a meta-analysis as a model for lepidopteran insect species, and exploring medical research using B. mori as a model for disease species by utilising transcriptome data., (© 2024. The Author(s).)

The growing use of nanomaterials has sparked significant interest in assessing the insect toxicities of nanoparticles. The silkworm, as an economically important insect, serves as a promising model for studying how insects respond to harmful substances. Here, we conducted a comprehensive investigation on the impact of graphene oxide (GO) on silkworms using a combination of physiological and transcriptome analyses. GO can enter the midguts and posterior silk glands of silkworms. High GO concentrations (> 25 mg/L) significantly (P < 0.01) inhibited larval growth. Additionally, GO (> 5 mg/L) significantly reduced the cocooning rate, and GO (> 15 mg/L) hindered oviduct development and egg laying in silkworms. GO increased the reactive oxygen species content and regulated catalase activity, suggesting that it may affect insect growth by regulating reactive oxygen detoxification. The transcriptome data analysis showed that 35 metabolism-related genes and 20 ribosome biogenesis-related genes were differentially expressed in response to GO, and their expression levels were highly correlated. Finally, we propose that a Ribosome biogenesis-Metabolic signaling network is involved in responses to GO. The research provides a new perspective on the molecular responses of insects to GO., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shengxiang Zhang reports financial support was provided by China Agriculture Research System of MOF and MARA. If there are other authors, they 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 The Authors. Published by Elsevier Inc. All rights reserved.)

Bombyx mori L. (Lepidoptera: Bombycidae) nucleopolyhedrovirus (BmNPV) is a serious pathogen causing huge economic losses to sericulture. There is growing evidence that the gut microbiota of silkworms plays a critical role in shaping host responses and interactions with viral infection. However, little is known about the differences in the composition and diversity of intestinal microflora, especially with respect to silkworm strain differences and BmNPV infection-induced changes. Here, we aim to explore the differences between BmNPV-resistant strain A35 and susceptible strain P50 silkworm and the impact of BmNPV infection on intestinal microflora in different strains. The 16S rDNA sequencing analysis revealed that the fecal microbial populations were distinct between A35 and P50 and were significantly changed post BmNPV infection in both strains. Further analysis showed that the BmNPV-resistant strain silkworm possessed higher bacterial diversity than the susceptible strain, and BmNPV infection reduced the diversity of intestinal flora assessed by feces in both silkworm strains. In response to BmNPV infection, the abundance of Muribaculaceae increased in P50 and decreased in A35, while the abundance of Enterobacteriaceae decreased in P50 and increased in A35. These results indicated that BmNPV infection had various effects on the abundance of fecal microflora in different silkworm strains. Our findings not only broadened the understanding of host-pathogen interactions but also provided theoretical help for the breeding of resistant strains and healthy rearing of silkworms based on symbiotic bacteria., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Paired box (Pax) genes are highly conserved throughout evolution, and the Pax protein is an important transcription factor of embryonic development. The Pax gene Bmgsb is expressed in the silk glands of silkworm, but its biological functions remain unclear. This study aimed to investigate the expression pattern of Bmgsb in the silk gland and explore its functions using RNA interference (RNAi). Here, we identified eight Pax genes in Bombyx mori. Phylogenetic analysis showed that the B. mori Pax genes were highly homologous to the Pax genes in other insects and highly evolutionarily conserved. The tissue expression profile showed that Bmgsb was expressed in the anterior silk gland and anterior part of the middle silk gland (AMSG). RNAi of Bmgsb resulted in defective development of the AMSG, and the larvae were mostly unable to cocoon in the wandering stage. RNA-seq analysis showed that the fibroin genes fib-l, fib-h and p25, cellular heat shock response-related genes and phenol oxidase genes were considerably upregulated upon Bmgsb knockdown. Furthermore, quantitative reverse transcription-PCR results showed that the fibroin genes and ubiquitin proteolytic enzyme-related genes were significantly upregulated in the AMSG after Bmgsb knockdown. This study provides a foundation for future research on the biological functions of B. mori Pax genes. In addition, it demonstrates the important roles of Bmgsb in the transcriptional regulation of fibroin genes and silk gland development., (© 2024 The Royal Entomological Society.)

A new globoscinic acid derivative, aspertubin A (1) along with four known compounds, were obtained from the co-culture of Aspergillus tubingensis S1120 with red ginseng. The chemical structures of compounds were characterized by using spectroscopic methods, the calculated and experimental electronic circular dichroism. Panaxytriol (2) from red ginseng, and asperic acid (4) showed significant antifeedant effect with the antifeedant rates of 75 % and 80 % at the concentrations of 50 μg/cm 2 . Monomeric carviolin (3) and asperazine (5) displayed weak attractant activity on silkworm. All compounds were assayed for antifungal activities against phytopathogens A. tubingensis, Nigrospora oryzae and Phoma herbarum and the results indicated that autotoxic aspertubin A (1) and panaxytriol (2) possessed selective inhibition against A. tubingensis with MIC values at 8 μg/mL. The co-culture extract showed higher antifeedant and antifungal activities against P. herbarum than those of monoculture of A. tubingensis in ordinary medium. So the medicinal plant and endophyte showed synergistic effect on the plant disease resistance by active compounds from the coculture of A. tubingensis S1120 and red ginseng., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

Scavenger receptor is pattern-recognition receptor (PRR) that plays a crucial function in host defense against pathogens. Scavenger receptor C (SR-C) is present only in invertebrates and its function has not been studied in detail. In this study, an SR-C homologous gene from the silkworm, Bombyx mori, was identified and characterized. SR-C was largely expressed in hemocytes and Malpighian tubules, with continuous expression in hemocytes. The peak expression was observed in hemocytes during molting and wandering stages both at mRNA and protein levels. Furthermore, immunofluorescence demonstrated it to be mainly distributed in the cell membranes of hemocytes, including oenocytoids and granulocytes. The recombinant SR-C protein (rSR-C) could bind to different types of bacteria and pathogen-associated molecular patterns (PAMPs), with strong binding to gram-positive bacteria and Lys-type peptidoglycans. The overexpression of SR-C induced the expression of genes related to the Toll pathway and antibacterial peptides. While the knockdown of SR-C reduced the expression of AMPs and inhibited the Toll pathway, it impaired the bacterial clearance ability of silkworm larvae, thus decreasing silkworm larvae's survival rate. Altogether, SR-C is a PRR that protect silkworms against bacterial pathogens by enhancing the expression of AMPs expression via the Toll pathway in hemocytes., (Copyright © 2021. Published by Elsevier B.V.)

The silkworm (Bombyx mori) is an important economic insect that can be used as food in many countries in Asia. However, silkworms and their metabolites are an important source of allergens, which can induce severe allergic reactions. So far, there are no systematic studies on the potential allergens in silkworm and its metabolites. These studies have important guiding significance for the prevention, diagnosis, and treatment of silkworm allergy. The aim of this study was to identify the potential allergens from larva, pupa, moth, silk, slough and feces of silkworm and analyze the sequence homology of silkworm allergens with other allergens identified in the Allergenonline database. We have found 45 potential allergens in silkworm. The results of the homology comparison suggested that silkworm allergens likely cross-react with those of Dermatophagoides farinae, Aedes aegypti, Tyrophagus putrescentiae, Triticum aestivum and Malassezia furfur., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Spider silk is one of the best natural fibers with excellent mechanical properties; however, due to the visual awareness, biting behavior and territory consciousness of spiders, we cannot obtain spider silk by large-scale breeding. Silkworms have a spinning system similar to that of spiders, and the use of transgenic technology in Bombyx mori, which is an ideal reactor for producing spider silk, is routine. In this study, the piggyBac transposon technique was used to achieve specific expression of two putative spider silk genes in the posterior silk glands of silkworms: aggregate spider glue 1 (ASG1) of Trichonephila clavipes (approximately 1.2 kb) and two repetitive units of pyriform spidroin 1 (PySp1) of Argiope argentata (approximately 1.4 kb). Then, two reconstituted spider silk-producing strains, the AG and PA strains, were obtained. Finally, the toughness of the silk fiber was increased by up to 91.5% and the maximum stress was enhanced by 36.9% in PA, and the respective properties in AG were increased by 21.0% and 34.2%. In summary, these two spider genes significantly enhanced the mechanical properties of silk fiber, which can provide a basis for spidroin silk production., (© 2021. The Author(s).)

Recent identification of a Piwi-interacting RNA (piRNA)-initiated sex determination cascade in the silkworm, Bombyx mori, provides novel insights into high diversity of insect sex determination pathways. In this system, the W-chromosome-derived Fem piRNA is the primary sex determination signal. A CCCH-type zinc finger gene Masculinizer (Masc), which is targeted by Fem piRNA-PIWI complex in female animals, is indispensable for male-specific splicing of B. mori doublesex (Bmdsx). Although many genes involved in this cascade have been identified, the regulatory mechanisms of silkworm sex determination remain to be elucidated. Here we show that another CCCH-type zinc finger gene, Bmznf-2, is a masculinization factor in B. mori. Bmznf-2 shows testis-abundant expression and loss of Bmznf-2 function via clustered regularly interspaced short palindromic repeats / single-guide RNA-mediated mutagenesis results in feminized differentiation and appearance of the female-specific splicing variants of Bmdsx transcripts in males. In contrast, there is no phenotypic consequence in mutant females. In mutant males, relative messenger RNA expression levels of female-dominant genes such as vitellogenin and sex-specific storage protein 1 are significantly elevated while several male-dominant genes are significantly down-regulated. Furthermore, male mutants show delayed developmental timing, smaller body sizes of larvae and malformation of moth wings. Our data thus reveal that Bmznf-2 plays an indispensable role in silkworm male sexual differentiation., (© 2020 Institute of Zoology, Chinese Academy of Sciences.)

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.)

The brown egg 4 (b-4) is a recessive mutant in the silkworm (Bombyx mori), whose egg and adult compound eyes exhibit a reddish-brown color instead of normal purple and black, respectively. By double digest restriction-site associated DNA sequencing (ddRAD-seq) analysis, we narrowed down a region linked to the b-4 phenotype to approximately 1.1 Mb that contains 69 predicted gene models. RNA-seq analysis in a b-4 strain indicated that one of the candidate genes had a different transcription start site, which generates a short open reading frame. We also found that exon skipping was induced in the same gene due to an insertion of a transposable element in other two b-4 mutant strains. This gene encoded a putative amino acid transporter that belongs to the β-group of solute carrier (SLC) family and is orthologous to Drosophila eye color mutant gene, mahogany (mah). Accordingly, we named this gene Bmmah. We performed CRISPR/Cas9-mediated gene knockout targeting Bmmah. Several adult moths in generation 0 (G 0 ) had totally or partially reddish-brown compound eyes. We also established three Bmmah knockout strains, all of which exhibit reddish-brown eggs and adult compound eyes. Furthermore, eggs from complementation crosses between the b-4 mutants and the Bmmah knockout mutants also exhibited reddish-brown color, which was similar to the b-4 mutant eggs, indicating that Bmmah is responsible for the b-4 phenotypes., (Copyright © 2021. Published by Elsevier Ltd.)

Silkworm, as a model organism, has very high economic value due to its silk secretion ability. Although a large number of studies have attempted to elucidate the mechanism of silk secretion, it remains unclear. In this study, the fibroin light chain (Fib-L) gene of silkworm was subjected to CRISPR/Cas9 editing, which yielded premature termination of translation at 135 aa. Compared with those of the wild type, the posterior silk glands (PSGs) of the homozygous mutants on the third day of the fifth instar showed obvious premature degeneration. Comparative transcriptome and proteomic analyses of the PSGs of wild-type individuals, heterozygous mutants and homozygous mutants were performed on the fourth day of the fifth instar. A GO enrichment analysis showed that the differentially expressed genes (DEGs) between homozygous mutants and wild-type individuals were enriched in cytoskeleton-related terms, and a KEGG enrichment analysis showed that the upregulated DEGs between homozygous mutants and wild-type individuals were enriched in the phagosome and apoptosis pathways. These results indicated that apoptosis was activated prematurely in the PSGs of homozygous mutants. Furthermore, autophagy and heat shock response were activated in the PSGs of homozygous mutants, as demonstrated by an analysis of the DEGs related to autophagy and heat shock. A comparative proteomic analysis further confirmed that autophagy, apoptosis and the heat shock response were activated in the PSGs of homozygous mutants, which led to premature degradation of the PSGs. These results provide insights for obtaining a more in-depth understanding of the mechanism of silk secretion in silkworms., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Cuticular proteins analogous to peritrophin 3 (CPAP3)-type cuticle proteins constitute a family of proteins with three chitin-binding domains (CBDs) that play an important role in cuticle formation by associating with chitin. In our previous study, we identified CPAP3-type cuticle proteins in the silkworm genome, of which we characterized CPAP3-A2 (BmCBP1), a protein highly expressed in the epidermis. In this study, to elucidate the digestion mechanism of CPAP3-type cuticle proteins, we incubated CPAP3-A2 with molting fluid in vitro and found that its hydrolysis, which was inhibited by serine and cysteine protease inhibitors, produced two major bands with a molecular weight of approximately 22 kD and 11 kD. A trypsin-type serine protease, p37k, was presumed to be responsible for hydrolyzing CPAP3-A2 based on liquid chromatography-tandem mass spectrometry analysis of naturally purified molting fluid. To verify this, p37k was subsequently expressed in Sf9 cells using the Bac-to-Bac baculovirus expression system. In its active form, the recombinant protease could successfully hydrolyze CPAP3-A2. Finally, we analyzed the CPAP3-A2 molting fluid digestion site. When arginine 169 of CPAP3-A2 was mutated to alanine, a weaker hydrolysis of mutant CPAP3-A2 was observed compared to that of normal CPAP3-A2. Collectively, we identified a trypsin-type serine protease that is involved in the degradation of CPAP3-type cuticle proteins, including CPAP3-A2, suggesting that this protease plays an important role during molting in Bombyx mori. These findings provide the basis for further elucidation of the mechanisms underlying insect molting and metamorphosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The insect brain is the central part of the neurosecretory system, which controls morphology, physiology, and behavior during the insect's lifecycle. Lepidoptera are holometabolous insects, and their brains develop during the larval period and metamorphosis into the adult form. As the only fully domesticated insect, the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process. Hormonal regulation in insects is a key factor in multiple processes. However, how juvenile hormone (JH) signals regulate brain development in Lepidoptera species, especially in the larval stage, remains elusive. We recently identified the JH receptor Methoprene tolerant 1 ( Met1 ) as a putative domestication gene. How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin's theory on domestication. Here, CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain, unlike precocious pupation of the cuticle. At the whole transcriptome level, the ecdysteroid (20-hydroxyecdysone, 20E) signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain. Pathways related to cell proliferation and specialization processes, such as extracellular matrix (ECM)-receptor interaction and tyrosine metabolism pathways, were suppressed in the brain. Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B . mori , which decreased transcriptional binding activity. The B. mori MET1 protein showed a changed structure and dynamic features, as well as a weakened co-expression gene network, compared with B . mandarina . Based on comparative transcriptomic analyses, we proposed a pathway downstream of JH signaling (i.e., tyrosine metabolism pathway) that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.

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.)

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.)

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusion of the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity, including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Vero cells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production of trimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture, and the underlying antiviral mechanism in silkworm is still unclear. Bombyx mori Nedd2-like caspase (BmNc) has been identified as a candidate antiviral gene from previous transcriptome data, since it is differentially expressed in the midgut of differentially resistant silkworm strains following BmNPV infection. However, the molecular mechanism by which BmNc responds to BmNPV is unknown. In this study, the relationship between BmNc and BmNPV was confirmed by its significantly different expression in different tissues of differentially resistant strains after BmNPV infection. Moreover, the antiviral role of BmNc was confirmed by the significantly higher fluorescence signals of BV-eGFP after knockdown of BmNc in BmN cells, and a reduced signal after overexpression. This was further verified by the capsid gene vp39 expression, DNA copy number, and GP64 protein level in the RNAi and overexpression groups. Furthermore, the antiviral phenomenon of BmNc was found to be associated with apoptosis. In brief, BmNc showed a relatively high expression level in the metamorphosis stages, and the effect of BmNc on BmNPV infection following RNAi and overexpression was eliminated after treatment with the inducer, Silvestrol, and the inhibitor, Z-DEVD-FMK, respectively. Therefore, it is reasonable to conclude that BmNc is involved in anti-BmNPV infection via the mitochondrial apoptosis pathway. The results provide valuable information for elucidating the molecular mechanism of silkworm resistance to BmNPV infection., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The single-celled pathogen Nosema bombycis, that can infect silkworm Bombyx mori and other lepidoptera including Spodoptera, is the first identified Microsporidia which has diplokaryotic nuclei throughout the life cycle. Septin proteins can form highly ordered filaments, bundles or ring structures related to the cytokinesis in fungi. Here, three septin proteins (NbSeptin1, NbSeptin2 and NbSeptin3) from Nosema bombycis CQ I are described. These proteins, appear to be conserved within the phylum Microsporidia. NbSeptins transcripts were detected throughout the pathogen developmental cycle and were significantly enhanced from second days of infection, which lead to our hypothesis that NbSeptins play a role in merogony. Immunofluorescence assay (IFA) revealed a broad distribution of NbSeptins in meronts and partly co-localization of NbSeptins. Interestingly, in some of meronts, NbSeptin2 and NbSeptin3 showed localization between the nuclei of the diplokaryon. Yeast two-hybrid and co-immunoprecipitation analysis verified that NbSeptins can interact with each other. Our findings suggest that NbSeptins can cooperate in the proliferation stage of Nosema bombycis and contribute towards the understanding of the rols of septins in microsporidia development., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Baculoviruses are classic pathogens that alter host behavior to enhance their dispersal and transmission. While viral protein tyrosine phosphatase (ptp) has been considered as a critical factor for inducing enhanced locomotory activity, preceding investigations have reported that viral ecdysteroid UDP-glucosyltransferase (egt) contributes to triggering climbing behavior in some virus and host species. Here we found that both egt and ptp were dispensable for these abnormal behaviors in Bombyx mandarina larvae induced by Bombyx mori nucleopolyhedrovirus, thus implying that there is an unknown core mechanism of baculovirus-induced alteration of host behaviors., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Root-knot nematodes cause damage to several crops and the importance of each species can vary according with the crop and the agricultural region. In Brazil, Meloidogyne javanica is one of the most important nematode species parasitizing mulberry. To define management strategies, it is important to know if the crop species is damaged by the parasitism of the nematode and the best choices for control, as the use of nematicides. Biological nematicides have been extensively used in Brazil, but no information regarding its efficiency to control M. javanica in mulberry is available. Besides, it is not known if biological nematicides could improve the quality of leaves or if they alter the nutrient composition of leaves, which could interfere in the development of the silkworms that are feed with these leaves or in the quality of the silk produced. With the aim to address these questions, we propose a study that will start in the phenotyping of the main Brazilian mulberry cultivars to Meloidogyne species, passing through the test of efficiency of biological nematicides in the control of M. javanica in mulberry cultivar Miura, evaluation of the amount and quality of leaves produced and, using these leaves to feed silkworms, in the analyzes of the impact of these diet in the health of silkworms, and in the production and quality of the silk., Competing Interests: No authors have competing interests.

Recent studies have demonstrated silk fibroin protein's (SF) ability to extend the shelf life of foods by mitigating the hallmarks of spoilage, namely oxidation and dehydration. Due to the potential for this protein to become more widespread, its safety was evaluated comprehensively. First, a bacterial reverse mutation test (Ames test) was conducted in five bacterial strains. Second, an in vivo erythrocyte test was conducted with Sprague Dawley rats at doses up to 1,000mg/kg-bw/day. Third, a range-finder study was conducted with Sprague Dawley rats at the highest consumption amount given solubility and oral gavage volume constrains (500mg/kg-bw/day). Fourth, a 28-day sub-chronic study in Sprague Dawley rats was conducted with the high dose set at 500mg/kg-bw/day, as limited by solubility of the protein in a single-gavage per-day study. Fifth, an in vitro pepsin digestion assay was performed to assess the potential for protein allergenicity. Sixth, allergenic potential was further assessed using liquid chromatography-mass spectroscopy for detection of allergenic insect proteins. Seventh, the SF protein sequences were subjected to bioinformatic analyses. Together, these studies raise no mutagenic, genotoxic, toxicological, or allergenic concerns with the oral consumption of silk fibroin., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The expression levels of some intrinsic genes, protease activity, and regulation of signaling pathways were distinct during different growth and development stages in the silkworm, Bombyx mori. The silkworm mutant mini was discovered from the normal silkworm strain S8V, and the body-size of the mini mutant was smaller than the wild-type from the second-instar and the difference became more significant in the following stages. In this study, genetic analysis of mini mutant showed that mini mutant was controlled by a single recessive gene, manifested as homozygous lethal. Then, the transcriptome analysis of the mini mutant indicated that 2944 differentially expressed genes (DEGs) were identified from the silkworm in the 48 h of the second-instar, of which 1638 genes in the mini mutants were upregulated and 1306 genes were downregulated. These DEGs were mainly distributed in the biological process, cellular component, and molecular function. The functional annotation based on the KEGG database showed that these genes were mainly clustered in metabolic pathways, fatty acid metabolism pathways, ribosome biogenesis in eukaryotes, and so on. Further analysis indicated that some genes involved in the growth and metabolism including enzyme genes, juvenile hormone, and ecdysone exhibited different transcriptional levels. These results provided new experimental evidence regarding the mechanism of the underlying formation of mini mutants., (© 2021 Wiley Periodicals LLC.)

Vitamin C (VC) is an essential nutrient for many animals. However, whether insects, including Bombyx mori, can synthesize VC remains unclear. In this article, the optimized HPLC method was used to determine the content of l-ascorbic acid (AsA) in silkworm eggs, larvae and pupae, and the activity of l-gulono-1,4-lactone oxidase (GULO), a key enzyme in VC synthesis. The RNA interference method was used to determine the effect of the BmGulo-like gene on embryonic development and GULO activity in the pupal fat body. The AsA content increased significantly during E144 h-E168 h in the late embryonic stage and P48 h-P144 h in the middle-late pupal stage, in which exogenous VC was not ingested. Furthermore, the body AsA content in larvae fed VC-free feed also increased with larval stage. The GULO enzymatic activity was present in eggs and the fat bodies of larvae and pupae, even when the larvae were reared with fresh mulberry leaves. Moreover, the activity was higher in the later embryonic stages (E144 h-E168 h) and the early pupal stage (before P24 h). The GULO activity in the pupal fat body dramatically decreased when the screened BmGulo-like gene (BGIBMGA005735) was knocked down with small interfering RNA; in addition, the survival rate and hatching rate of eggs significantly decreased 21% and 44%, respectively, and embryonic development was delayed. Thus, Bombyx mori can synthesize AsA through the l-gulose pathway, albeit with low activity, and this synthesis ability varies with developmental stages., (© 2021 Wiley Periodicals LLC.)

Lactic acid is widely used in the food, drugs, cosmetics, and other industries to maintain the microbial stability of low-pH products. However, it is unclear whether lactic acid can affect silkworm (Bombyx mori) growth and reproduction. This study investigated the effects of lactic acid on the growth and reproduction of the silkworm. We analyzed the growth, cocoon quality, and reproductive performance of fifth instar larvae fed on mulberry leaves saturated with different concentrations (0.01, 0.1, 1, and 10%) of lactic acid and the control. Results showed that 0.01, 0.1, and 1% lactic acid supplementation positively affects growth and female cocoon quality, with increased larval weight and female cocoon shell weight compared to the control group. In contrast, 10% lactic acid was toxic to the larvae and significantly decreased growth, leading to larval death. Our study provides a basic reference for the optimal amount of preservatives. In addition, this study can be a desirable intervention for sericulturists and can play an important role in getting high return from silkworm-rearing activities., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.)

Background: Krüppel homolog 1 (Kr-h1) is a critical transcription factor for juvenile hormone (JH) signaling, known to play a key role in regulating metamorphosis and adult reproduction in insects. Kr-h1 can also be induced by molting hormone 20-hydroxyecdysone (20E), however, the underlying mechanism of 20E-induced Kr-h1 expression remains unclear. In the present study, we investigated the molecular mechanism of Kr-h1 induction by 20E in the reproductive system of a model lepidopteran insect, Bombyx mori., Results: Developmental and tissue-specific expression analysis revealed that BmKr-h1 was highly expressed in ovaries during the late pupal and adult stages and the expression was induced by 20E. RNA interference (RNAi)-mediated depletion of BmKr-h1 in female pupae severely repressed the transcription of vitellogenin receptor (VgR), resulting in the reduction in vitellogenin (Vg) deposition in oocytes. BmKr-h1 specifically bound the Kr-h1 binding site (KBS) between - 2818 and - 2805 nt upstream of BmVgR and enhanced the transcription of BmVgR. A 20E cis-regulatory element (CRE) was identified in the promoter of BmKr-h1 and functionally verified using luciferase reporter assay, EMSA and DNA-ChIP. Using pull-down assays, we identified a novel transcription factor B. mori Kr-h1 regulatory protein (BmKRP) that specifically bound the BmKr-h1 CRE and activated its transcription. CRISPR/Cas9-mediated knockout of BmKRP in female pupae suppressed the transcription of BmKr-h1 and BmVgR, resulting in arrested oogenesis., Conclusion: We identified BmKRP as a new transcription factor mediating 20E regulation of B. mori oogenesis. Our data suggests that induction of BmKRP by 20E regulates BmKr-h1 expression, which in turn induces BmVgR expression to facilitate Vg uptake and oogenesis.

In insects, sex determination pathways involve three levels of master regulators: primary signals, which determine the sex; executors, which control sex-specific differentiation of tissues and organs; and transducers, which link the primary signals to the executors. The primary signals differ widely among insect species. In Diptera alone, several unrelated primary sex determiners have been identified. However, the doublesex ( dsx ) gene is highly conserved as the executor component across multiple insect orders. The transducer level shows an intermediate level of conservation. In many, but not all examined insects, a key transducer role is performed by transformer ( tra ), which controls sex-specific splicing of dsx . In Lepidoptera, studies of sex determination have focused on the lepidopteran model species Bombyx mori (the silkworm). In B. mori, the primary signal of sex determination cascade starts from Fem , a female-specific PIWI-interacting RNA, and its targeting gene Masc , which is apparently specific to and conserved among Lepidoptera. Tra has not been found in Lepidoptera. Instead, the B. mori PSI protein binds directly to dsx pre-mRNA and regulates its alternative splicing to produce male- and female-specific transcripts. Despite this basic understanding of the molecular mechanisms underlying sex determination, the links among the primary signals, transducers and executors remain largely unknown in Lepidoptera. In this review, we focus on the latest findings regarding the functions and working mechanisms of genes involved in feminization and masculinization in Lepidoptera and discuss directions for future research of sex determination in the silkworm.

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.)

The Janus kinase/signal transducer and activator of transcription cascade transduction (JAK/STAT) signaling pathway is highly conserved in mammals, but the pattern recognition receptors (PRRs) and their functions are unclear. We found that the expression pattern of Bombyx mori C-type lectin 5 (BmCTL 5) had a synergy relevance with the JAK/STAT signaling pathway against Beauveria bassiana. An RNAi assay, subcellular localization analysis, yeast two-hybrid technique, protein recruitment experiment and pathogen infection tests were used to explore the roles of BmCTL 5 in the JAK/STAT signaling pathway. Knock-down of the BmCTL 5 suppressed the JAK/STAT signaling pathway and the PO cascade of nodule melanization. BmCTL 5 is located in the cytomembrane and interacted with BmHOP both in yeast and B. mori ovary cells N (BmN cells). BmCTL 5 and the JAK/STAT signaling pathway was activated by B. bassiana but only slightly activated by B. mori cytoplasmic polyhedrosis virus (BmCPV), Nosema bombycis and bacteria LPS. These findings suggest that BmCTL 5 might be an important PRR for the JAK/STAT signaling pathway and may mediate the nodule melanization for fungi infection. These data provide insights into the immune mechanism of the JAK/STAT signaling pathway in insects and aid understanding of the mechanism of the JAK/STAT signaling pathway and adaptive immune systems in mammals., (Copyright © 2020. Published by Elsevier Inc.)

Krüppel homolog 1 (Kr-h1), a zinc finger transcription factor, is involved in the metamorphosis and adult reproduction of insects. However, the role of Kr-h1 in reproduction of holometabolic insects remains to be elucidated. The regulation network of Kr-h1-associated genes in the reproduction in Bombyx mori was investigated in this study. The higher expression level of BmKr-h1 in the ovaries was detected during the late pupal stage and adults. RNA interference (RNAi)-mediated depletion of BmKr-h1 in the female at day 6 of pupae resulted in abnormal oocytes at 48 h post-double-stranded RNA treatment, which showed less yolk protein deposition and partially transparent chorion. RNA-seq and subsequent differentially expressed transcripts analysis showed that knockdown of BmKr-h1 caused a decrease in the expression of 2882 genes and an increase in the expression of 2565 genes in the oocytes at day 8 of pupae. Totally, 27 genes coding for transcription factors were down-regulated, while six genes coding for other transcription factors were up-regulated. BmKr-h1 bound to the Kr-h1 binding site of the transcription factors AP-1 (activating protein-1) and FOXG1 to increase their messenger RNA transcripts in the BmN cells, respectively. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses of that positively co-expressed with AP-1 and FOXG1 transcripts showed mainly enrichment in the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes. These data suggested that BmKr-h1 might directly regulate the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes or probably through AP-1 and /or FOXG1 to regulate oocyte development., (© 2020 Institute of Zoology, Chinese Academy of Sciences.)

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of primary silkworm pathogens and causes a serious damage of cocoon losses every year. Recent years, many works have been done to clarify the silkworm anti-BmNPV mechanism, and a significant progress has been made in screening and studying of genes and proteins related to BmNPV infection, but several of them lacked the proofs in vivo. In this study, to further validate the function of seven newly reported genes in vivo, including BmAtlatin-n, Bmferritin-heavy chain (BmFerHCH), Bmthymosin (BmTHY), Bmseroin1, Bmseroin2, Bmnuclear hormone receptors 96 (BmNHR96), and BmE3 ubiquitin-protein ligase SINA-like 10 (BmSINAL10), the response of them in the midgut, fat body, and hemolymph of differentially resistant strains (resistant strain YeA and susceptible strain YeB) at 48 h following BmNPV infection were analyzed. The results showed that the relative stable or upregulated expression level of BmAtlatin-n, BmTHY, Bmseroin1, and Bmseroin2 in YeA resistant strain following BmNPV infection further indicated their antiviral role in vivo, compared with susceptible YeB strain. Moreover, the significant downregulation of BmFerHCH, BmNHR96, and BmSINAL10 in both strains following BmNPV infection revealed their role in benefiting virus infection, as well as the upregulation of BmFerHCH in YeB midgut and BmSINAL10 in YeB hemolymph. These data could be used to complementary the proofs of the function of these genes in response to BmNPV infection., (© 2020 Wiley Periodicals LLC.)

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.)