Identification of function modules in the co-expression protein-protein interaction network of Bombyx mori in response to Beauveria bassiana infection.

Beauveria bassiana (B. bassiana) is a common fungal disease in sericulture. Previous research has primarily focused on investigating genes involved in innate immunity. However, the response of Bombyx mori (B. mori) to B. bassiana requires the coordination of other biological processes in addition to the immune system. We measured protein expression profile of B. mori after inoculating B. bassiana using iTRAQ technology in previous. Here we constructed a co-expression protein-protein interaction network of B. mori in response to B. bassiana infection. Subnetworks and modules were analyzed, and the functions of these modules were annotated. The results revealed the identification of numerous proteins associated with cellular immunity, including those involved in phagosomes, lysosomes, mTOR signaling, sugar metabolism, and the ubiquitin-proteasome pathway. Meanwhile, we observed that the pathways involved in protein synthesis were activated, including pyruvate and purine metabolism, RNA transport, ribosome, protein processing in endoplasmic reticulum, and protein export pathways, during B. bassiana infection. Based on this analysis, we selected six candidate genes (shock protein, ribosome, translocon, actin muscle-type A2, peptidoglycan recognition protein, and collagenase) that were found to be related to the response to B. bassiana. Further verification experiments demonstrated significant changes in their expression levels after inoculation with B. bassiana. These research findings provide new insights into the molecular mechanism of insect immune response to fungal infection.

RNA-seq analysis of LPS-induced immune priming in silkworms (Bombyx mori) and the role of cytochrome P450 detoxification system in the process.

While immune priming has been identified in many invertebrates, the intricate mechanisms that drive this process in insects continue to be a subject of mystery. In this study, we exposed silkworm larvae to varying doses of lipopolysaccharide (LPS) to induce immune priming and assessed their survival upon challenge with Bacillus thuringiensis (Bt). Transcriptome analysis was performed to identify differentially expressed genes (DEGs) associated with immune priming. The role of CYP450 genes in this process was further explored using RNA interference (RNAi) to knockdown CYP9E2 and CYP6K1, followed by measurements of detoxification enzyme activities and reactive oxygen species (ROS) levels. We found that LPS exposure significantly increased silkworm survival rates upon Bt challenge, indicating the induction of immune priming. Transcriptome analysis revealed 549 DEGs, including a large number involved in detoxification, immunity, and metabolism, suggesting a complex regulatory network that encompasses immune responses and metabolic pathways. Functional enrichment and gene set enrichment analysis (GSEA) highlighted the activation of immune signaling pathways and the involvement of detoxification processes. Knockdown of CYP9E2 and CYP6K1 resulted in increased ROS levels, decreased detoxification enzyme activities, and reduced survival rates post-Bt challenge, implicating the critical role of these genes in immune priming and detoxification. Our findings demonstrate that LPS-induced immune priming in silkworms involves the upregulation of CYP450 genes, which play a critical role in detoxification and immune response modulation. The study provides insights into the molecular mechanisms of immune priming in insects and highlights the potential of CYP9E2 and CYP6K1 as targets for enhancing disease resistance and pest management in insects.

Exploring the structure and dynamics of soft and hard cuticle of Bombyx mori using solid-state NMR techniques.

This study conducts a comprehensive analysis and comparison of Bombyx mori cuticles across different developmental stages, ranging from larval to adult, utilizing advanced solid-state NMR techniques. The primary objective is to elucidate the underlying reasons for the contrasting hardness of adult cuticles and softness of larval cuticles. Notably, PXRD analysis reveals a prominent broad peak at 19.34°, indicating the predominantly amorphous nature of both larval and adult cuticles. Analysis of 13C CP-MAS SSNMR spectra highlights an elevated proportion of phenoxy carbon in adult cuticles (6.77%) compared to larval cuticles (1.24%). Furthermore, a distinctive resonance line at 144 ppm is exclusively observed in adult cuticles, due to catechols, suggesting potential biochemical pathway variations during development. Significant variations in the primary components of 13C chemical shift anisotropy (CSA) tensors for aliphatic carbons of amino acids, catechols, and lipids between adult and larval cuticles indicate alterations in electronic environments. Additionally, the shorter spin-lattice relaxation time of carbon nuclei in larval cuticles compared to adult cuticles implies slower motional dynamics with enhanced degree of sclerotization in adults. By investigating the internal structure and dynamics of cuticles, this research not only contributes to biomimetic material development but also enhances our understanding of structural changes across different developmental stages of B. mori.

BmToll9-1 Is a Positive Regulator of the Immune Response in the Silkworm Bombyx mori.

Toll receptors are involved in the development and innate immunity of insects. BmToll9-1 is an important immune receptor in the Toll pathway. Previous studies have focused on its role as a receptor in immune response. In this study, we aimed to investigate the role of BmToll9-1 as a regulator in the immune response. The expression profiles demonstrated that BmToll9-1 was predominantly expressed in the midgut. RNA interference (RNAi) of BmToll9-1 was found to be effective in the midgut via the injection of dsRNA, which resulted in smaller and lighter larvae and cocoons. Most signaling genes in the Toll pathway and downstream effector genes were downregulated after the RNAi of BmToll9-1. The hemolymph from BmToll9-1-silenced larvae showed decreased antibacterial activity against Escherichia coli, either in growth curve or inhibition zone experiments. The above results indicate that BmToll9-1 might be positively involved in the immune pathway of silkworm. As a positive regulator, BmToll9-1 might function mainly in the gut to maintain microbial homeostasis to regulate the growth of silkworms. Silencing of BmToll9-1 downregulates the signaling genes in the Toll pathway and antimicrobial peptide (AMP) production, resulting in decreased antibacterial activity in the hemolymph.

Transcriptome analysis provides new insight into the mechanism of Bombyx mori under zinc exposure.

Zinc is a significant source of heavy metal pollution that poses risks to both human health and biodiversity. Excessive concentrations of zinc can hinder the growth and development of insects and trigger cell death through oxidative damage. The midgut is the main organ affected by exposure to heavy metals. The silkworm, a prominent insect species belonging to the Lepidoptera class and widely used in China, serves as a model for studying the genetic response to heavy metal stress. In this study, high-throughput sequencing technology was employed to investigate detoxification-related genes in the midgut that are induced by zinc exposure. A total of 11,320 unigenes and 14,723 transcripts were identified, with 553 differentially expressed genes (DEGs) detected, among which 394 were up-regulated and 159 were down-regulated. The Gene Ontology (GO) analysis revealed that 452 DEGs were involved in 18 biological process subclasses, 14 cellular component subclasses and 8 molecular functional subclasses. Furthermore, the KEGG analysis demonstrated enrichment in pathways such as Protein digestion, absorption and Lysosome. Validation of the expression levels of 9 detoxification-related DEGs through qRT-PCR confirmed the accuracy of the RNA-seq results. This study not only contributes new insights into the detoxification mechanisms mechanism of silkworms against zinc contamination, but also serves as a foundation basis for understanding the molecular detoxification processes in lepidopteran insects.

Identification of the oosporein biosynthesis gene cluster in an entomopathogenic fungus Blackwellomyces cardinalis.

Blackwellomyces cardinalis (≡ Cordyceps cardinalis) is an entomopathogenic fungus that hosts lepidopteran insect larvae. Oosporein, produced by Bl. cardinalis, is a red secondary metabolite that is also produced by other entomopathogens and is known to contribute to entomopathogenic activity. In this study, a homologous region of the oosporein biosynthesis gene cluster (BcOpS cluster) was found from the genome sequence of Bl. cardinalis strain NBRC 103832. Within the cluster, a putative transcription factor gene BcOpS3 was deleted by homologous recombination. The deletion strain (ΔBcOpS3) did not produce oosporein. Real-time qPCR analysis showed that the expression of all genes was either lost or greatly reduced compared to the wild type strain (WT). Infection assay using silkworms showed that the virulence of the ΔBcOpS3 strain was not different from that of the WT strain. We compared the expression levels of antimicrobial peptide genes in silkworm infected with these strains, and found that the increased expression of the cecA gene in WT was not observed in the ΔBcOpS3 strain, suggesting that the immune response of the silkworm was altered.

The mechanical properties of chimeric silk are improved by expressing the full-length Trichonephila clavipes major ampullate spidroin gene in the silkworm Bombyx mori via recombinant AcMNPV.

Spider silk is a type of natural protein fiber with excellent toughness and tensile strength. The mechanical properties of chimeric silk have been improved by integrating the spider silk protein gene into the silkworm (Bombyx mori) genome, but this strategy requires a long time to produce genetically modified silkworms. In this study, to rapidly produce chimeric silkworms/spider silk with improved toughness and tensile strength, recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV), AcMNPV-FHP-MaSp-G, harboring a full-length Trichonephila clavipes major ampullate spidroin G (MaSp-G) gene driven by the silkworm fibroin heavy chain (Fib-H) promoter, was constructed, in which the signal peptide sequence of the MaSp-G gene was replaced by the signal peptide sequence of the Fib-H gene. Western blot and LC-MS/MS results showed that MaSp-G was successfully expressed in the posterior silk gland of silkworm larvae infected with AcMNPV-FHP-MaSp-G and secreted into the cocoon. Mechanical property tests revealed that the average maximum breaking stress and the average maximum elastic strain of chimeric silkworms/spider silk were 497.867 MPa and 14.824%, respectively, which were 36.53% and 23.55% greater than those of silk produced by normal silkworms. Fourier transform infrared (FTIR) spectroscopy revealed that the proportions of ß-sheets, α-helices, and ß-turns in the chimeric silk increased by 18.22%, 16.92%, and 18.72%, respectively. These results indicate that the mechanical properties of the chimeric silk produced by silkworms infected with AcMNPV-FHP-MaSp-G were significantly improved, which provides a new method for rapid production of chimeric silk in a genetically modified/genome-edited silkworm-independent manner.

Molecular Functions and Physiological Roles of Gustatory Receptors of the Silkworm Bombyx mori.

Complete elucidation of members of the gustatory receptor (Gr) family in lepidopteran insects began in the silkworm Bombyx mori. Grs of lepidopteran insects were initially classified into four subfamilies based on the results of phylogenetic studies and analyses of a few ligands. However, with further ligand analysis, it has become clear that plant secondary metabolites are important targets not only for Grs in the bitter subfamily but also for the Drosophila melanogaster Gr43a orthologue subfamily and Grs in the sugar subfamily. Gene knockout experiments showed that B. mori Gr6 (BmGr6) and BmGr9 are involved in the recognition of the feeding-promoting compounds chlorogenic acid and isoquercetin in mulberry leaves by the maxillary palps, suggesting that these Grs are responsible for palpation-dependent host recognition without biting. On the other hand, BmGr expression was also confirmed in nonsensory organs. Midgut enteroendocrine cells that produce specific neuropeptides were shown to express specific BmGrs, suggesting that BmGrs are involved in the induction of endocrine secretion in response to changes in the midgut contents. Furthermore, gene knockout experiments indicated that BmGr6 is indeed involved in the secretion of myosuppressin. On the other hand, BmGr9 was shown to induce signal transduction that is not derived from the intracellular signaling cascade mediated by G proteins but from the fructose-regulated cation channel of BmGr9 itself. Cryogenic electron microscopy revealed the mechanism by which the ion channel of the BmGr9 homotetramer opens upon binding of fructose to the ligand-binding pocket. Research on BmGrs has contributed greatly to our understanding of the functions and roles of Grs in insects.

Selenium Treatment Ameliorates the Adverse Effects Caused by Dynamin Gene Knockdown in Bombyx mori.

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.

Bombyx mori serpin 3 is involved in innate immunity by interacting with serine protease 7 to regulate prophenoloxidase activation.

A subfamily of conserved proteins called serpins plays crucial roles in various physiological functions, particularly in the activation pathway of the serine protease cascade, an essential component of insect innate immunity. Here, we found Bombyx mori serpin 3 (BmSerpin3) was most highly expressed in the fat body, and was up-regulated after exposure to bacteria, fungus and virus. Further, the expression of BmSerpin3 in the hemocytes, fat body, midgut of silkworm larvae, and BmN cells was up-regulated upon Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Through Bac-to-Bac expression system, we obtained the active protein of BmSerpin3, and the enzyme activity assay showed that BmSerpin3 significantly inhibited the activity of both subtilisin and trypsin. In addition, BmSerpin3 could inhibit the activation of prophenoloxidase (PPO) in larvae. The knockdown of BmSerpin3 showed increased phenoloxidase (PO) activity compared to control after BmNPV infection. Ultimately, we confirmed that BmSerpin3 interacts with B. mori Serine Protease 7 (BmSP7). Hence, we hypothesize that BmSerpin3 is involved in innate immunity by interacting with BmSP7 to regulate the PPO activation cascade. Taken together, these results showed that BmSerpin3 play a role in silkworm innate immunity and lay a foundation for studying its functions.

The development of silk glands and transcriptome aberration induced by cyantraniliprole in Bombyx mori.

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 LC5, LC10 and LC20, 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 LC5 treatment. Histopathological and ultrastructural analysis revealed that Cya LC10 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 LC10, 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 LC10 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.

Soluble Guanylate Cyclase α1 Gene Influences Egg-Laying Amount and Hatching Rate in Bombyx mori.

Soluble guanylate cyclase (sGC) serves as a receptor of nitric oxide (NO) and is the core metalloenzyme in the NO signal transduction pathway. sGC plays a key role in the NO-cGMP signal transduction pathway and participates in various physiological processes, including cell differentiation, neuron transmission, and internal environment homeostasis. sGC consists of two subunits, α and ß, each subunit containing multiple isoforms. In this study, we cloned and analyzed the sGC-α1 gene in the silkworm Bombyx mori (BmsGC-α1). The BmsGC-α1 gene was expressed highest at the pupal stages. The highest BmsGC-α1 mRNA expression was observed in the head of fifth instar larvae and in fat body during the wandering stage of B. mori. Furthermore, we observed that feeding fifth instar larvae with thyroid hormone and nitroglycerin induced the expression of the BmsGC-α1 gene. Injection of BmsGC-α1 siRNA into silkworms at the prepupal stage resulted in a significant decrease in BmsGC-α1 expression levels at 48 and 72 h postinjection. After silencing BmsGC-α1, both the egg-laying amount and hatching rate of silkworm eggs were significantly reduced compared to the control group. These results suggest that BmsGC-α1 plays an important role in regulating the reproductive system of silkworms. This finding enhances our understanding of the functional diversity of sGC in insects.

Translocator protein (TSPO) inhibits Nosema bombycis proliferation in silkworm, Bombyx mori.

Pebrine disease, caused by Nosema bombycis (Nb) infection in silkworms, is a severe and long-standing disease that threatens sericulture. As parasitic pathogens, a complex relationship exists between microsporidia and their hosts at the mitochondrial level. Previous studies have found that the translocator protein (TSPO) is involved in various biological functions, such as membrane potential regulation, mitochondrial autophagy, immune responses, calcium ion channel regulation, and cell apoptosis. In the present study, we found that TSPO expression in silkworms (BmTSPO) was upregulated following Nb infection, leading to an increase in cytoplasmic calcium, adenosine triphosphate, and reactive oxygen species levels. Knockdown and overexpression of BmTSPO resulted in the promotion and inhibition of Nb proliferation, respectively. We also demonstrated that the overexpression of BmTSPO promotes host cell apoptosis and significantly increases the expression of genes involved in the immune deficiency and Janus kinase-signal transducer and the activator of the transcription pathways. These findings suggest that BmTSPO activates the innate immune signalling pathway in silkworms to regulate Nb proliferation. Targeting TSPO represents a promising approach for the development of new treatments for microsporidian infections.

CRISPR/Cas9-mediated disruption of orf76 as an antiviral therapy against BmNPV in the transgenic silkworm.

Viral diseases pose a significant threat to livestock husbandry and plant cultivation. CRISPR/Cas9-mediated targeted editing of viral genes offers a promising approach to antiviral therapy. The silkworm, Bombyx mori, is an economically important insect susceptible to infection by B. mori nucleopolyhedrovirus (BmNPV), and viral outbreaks cause severe economic losses to the sericulture industry. Here, we identified BmNPV orf76 as a viral late gene that is highly similar to Autographa californica multiple nucleopolyhedrovirus Ac93. The deletion of orf76 abolished BmNPV proliferation and hindered the production of infectious budded viruses. We generated a transgenic line, Cas9(+)/sgorf76(+), that did not affect the growth or development of the silkworm and demonstrated that the transgenic line Cas9(+)/sgorf76(+) efficiently cleaved orf76 at the sgorf76 site, resulting in large deletions at 120 h post-infection, with no observed off-target effects. Survival analyses revealed that the transgenic line Cas9(+)/sgorf76(+) exhibited significantly higher survival rates than the control lines Cas9(-)/sgorf76(-), regardless of the BmNPV inoculation dose. Additionally, the number of BmNPV DNA copies and the expression levels of viral genes were markedly inhibited in the transgenic line Cas9(+)/sgorf76(+) compared with the control line Cas9(-)/sgorf76(-). The results provide a promising target for Cas9-mediated antiviral therapy against BmNPV, and the findings provide new insights for baculovirus gene function studies and lepidopteran pest control.

Effects of mulberry leaf enrichment with Lepidium sativum L. seed powder suspension on the economic parameters of Bombyx mori L.

The phytochemicals of high nutritional and functional properties in Lepidium sativum L. (garden cress) seeds have nominated their seed powder (regardless of the concentration used) for enrichment of mulberry leaves in order to enhance Bombyx mori L. larval feeding, and consequently to gain ground in sericulture industry. As expected, B. mori larval feeding on L. sativum-enriched mulberry leaves showed not only a remarkable increase in mean values of certain economic parameters of B. mori, such as cocoon weight, cocoon shell weight, pupal weight, and egg yield, compared with the control group, but also showed a phenomenal increase in egg counts (on average, ca. 958-1256 eggs laid per female moth) and a significant increase in egg size (measured as egg surface area and egg volume). Male or female moth larval diet has significantly influenced the reproductive performance or fitness of both sexes of B. mori in terms of large-sized moths (measured as forewing, hind femur, and hind tibia lengths) and highly fecund moths (i.e., increased fecundity and spermatophore counts per female moth, and large-sized eggs). On the basis of B. mori female moth reproductive index, the female moths from L. sativum-fed larvae proved to have a lower reproductive index compared to their corresponding value for females of the control group, indicating more efficient utilization of larval resources for B. mori reproduction. Quantification of the three main physiological resources viz., protein, lipid and carbohydrate in the internal reproductive tract of B. mori female moths at death has nominated the female moth abdomens, or simply their bodies, as being a reasonable natural source of protein, lipid, and carbohydrate, to be involved in certain manufactures (e.g., pet feed formulations) instead of discarding them as a source of environmental pollution. Evidently, the L. sativum seed powder is of considerable interest because it remarkably improves the performance of such an economically important insect, B. mori. This is the first study for evaluating the efficacy of L. sativum seed powder in sericulture field to enhance B. mori productivity parameters.

Evaluation of in vivo pharmacokinetic study of the anti-cancer drug imatinib using silkworms as an animal model.

Imatinib is an oral molecular targeted therapy that acts as a tyrosine kinase inhibitor. Silkworms present a promising experimental model for elucidating the pharmacokinetic and toxicity profiles of various compounds. This study aimed to establish an experimental paradigm for investigating the pharmacokinetics of imatinib in silkworms. A comparative analysis of imatinib pharmacokinetic parameters across silkworms, humans, mice, and rats revealed similarities in time to maximum concentration (Tmax) and apparent clearance values between silkworms and humans. However, differences in elimination half-life (t1/2) and apparent volume of distribution between silkworms and humans remained within 5- and 4-fold ranges, respectively. Importantly, mice demonstrated pharmacokinetic parameters closer to those of humans than rats during imatinib studies. Additionally, silkworms and mice exhibit similar Tmax and t1/2 values. This study highlights the potential of silkworms as valuable tools for investigating imatinib metabolism in pharmacokinetic studies. Furthermore, it underscores the applicability of silkworms in elucidating the pharmacokinetic parameters of various molecular-targeted drugs, thus facilitating advancements in drug development and evaluation.

Comparison of adipose derived stromal cells cultured on fibroin scaffolds fabricated by salt-leaching and by freeze-thawing.

Fibroin, the main structural protein of Bombyx mori silk, is known for its mechanical properties, its biocompatibility and degradation characteristics in vivo. Various studies investigate its uses as cell carrier and/or material for surgical implants. Multiple protocols have been established to isolate fibroin from silk fibers and to produce scaffolds and films from fibroin solution. There is only limited literature available on how fibroin scaffolds manufactured by different methods compare to each other in terms of performance as cell carriers. This study compares the behaviour of human adipose derived stromal cells (ADSC) seeded on fibroin scaffolds produced by (i) salt-leaching and (ii) freeze-thawing. One type of freeze-thawing scaffold (poresize â‰ª 315 µm) and three types of salt-leaching scaffolds (poresize ranging from 315 µm to 1000 µm) were used for this comparison. Measuring the DNA concentration on the seeded scaffolds as well as the seeded cells metabolic activity, we were able to determine freeze-thawed scaffolds to be superior for cell-seeding. ADSC seeded on salt-leaching scaffolds displayed a stronger downregulation of serum deprivation response gene than cells seeded on freeze-thaw scaffolds. In sum, our findings show that salt-leaching scaffolds offering different pore sizes differed much less among each other than salt-leaching from freeze-thawing scaffolds in terms of cell accommodation. Our work underlines the importance of physicochemical scaffold properties directly linked to different manufacturing methods and their influence on the cell seeding capacity of silk fibroin based carriers.

Insights into the activation mechanism of Bm-CPA: Implications for insect molting regulation.

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.

PI3K-Akt-SGF1-Dimm pathway mediates the nutritional regulation of silk protein synthesis in Bombyx mori.

The efficient synthesis of silk protein is heavily reliant on the ingestion of massive nutrients during the peak growth phase in the silkworm. However, the molecular mechanism of nutritional regulation of silk protein synthesis remains unknown. In this study, we investigated the impact of nutrient deficiency on the synthesis of silk protein. Nutritional deficiency led to a reduction in silk yield, accompanied by decreased levels of silk proteins and fibroin heavy chain (FibH)-activating transcription factors SGF1 and Dimm. Furthermore, insulin enhanced the protein levels of SGF1 and Dimm, which can be attenuated by specific inhibitors of PI3K. Co-immunoprecipitation analysis showed that the nutrient pathway factor protein kinase B (Akt) could interact with SGF1 protein. Knockdown of Akt reduced the phosphorylation level of SGF1 and impedes its nuclear translocation. Further studies revealed that SGF1 was directly bound to Fkh site in the 22-43 region upstream of ATG of Dimm gene to activate its transcription. In conclusion, during the peak growth phase, nutrition promotes the massive synthesis of silk protein through the PI3K-Akt-SGF1-Dimm pathway. This study offers valuable insights into the efficient synthesis of silk proteins and establishes a theoretical foundation for improving silk yield.

Bombyx mori nucleopolyhedrovirus LEF-2 disrupts the cell cycle in the G2/M phase by triggering a host cell DNA damage response.

It is a common strategy for viruses to block the host cell cycle to favour their DNA replication. Baculovirus, being a double-stranded DNA virus, can arrest the cell cycle in the G2/M phase to facilitate its replication. However, the key viral genes and mechanisms crucial for inducing cell cycle arrest remain poorly understood. Here, we initially examined the impacts of several Bombyx mori nucleopolyhedrovirus (BmNPV) DNA replication-associated genes: ie1, lef-1, lef-2, lef-3, lef-4, odv-ec27 and dbp. We assessed their effects on both the host cells' DNA replication and cell cycle. Our findings reveal that when the lef-2 gene was overexpressed, it led to a significant increase in the number of cells in the G2/M phase and a reduction in the number of cells in the S phase. Furthermore, we discovered that the LEF-2 protein is located in the virogenic stroma and confirmed its involvement in viral DNA replication. Additionally, by employing interference and overexpression experiments, we found that LEF-2 influences host cell DNA replication and blocks the cell cycle in the G2/M phase by regulating the expression of CyclinB and CDK1. Finally, we found that BmNPV lef-2 triggered a DNA damage response in the host cell, and inhibiting this response removed the cell cycle block caused by BmNPV LEF-2. Thus, our findings indicate that the BmNPV lef-2 gene plays a crucial role in viral DNA replication and can regulate host cell cycle processes. This study furthers our understanding of baculovirus-host cell interactions and provides new insight into the molecular mechanisms of antiviral research.