Search Results (50)

Background/Objective: Platelet concentrates (PCs) are used in transfusion medicine to treat bleeding disorders. Staphylococcus aureus, a predominant PC contaminant, has been implicated in several adverse transfusion reactions. The aim of this study was to investigate the impact of PC storage on S. aureus resistance to quinolones, which are commonly used to treat S. aureus infections. Methods/Results: Four transfusion-relevant S. aureus strains (TRSs) were subjected to comparative transcriptome analyses when grown in PCs vs. trypticase soy broth (TSB). Results of these analyses revealed differentially expressed genes involved in antibiotic resistance. Of interest, the norB gene (encodes for the NorB efflux pump, which is implicated in quinolone resistance and is negatively regulated by MgrA) was upregulated (1.2–4.7-fold increase) in all PC-grown TRS compared to TSB cultures. Minimal Bactericidal Concentration (MBC) of ciprofloxacin and norfloxacin in PC-grown TRS compared to TSB showed increased resistance to both quinolones in PC cultures. Complementary studies with non-transfusion-relevant strains S. aureus RN6390 and its norB and mgrA deletion mutants were conducted. MBC of ciprofloxacin and norfloxacin and RT-qPCR assays of these strains showed that not only norB, but also norA and norC may be involved in enhanced quinolone resistance in PC-grown S. aureus. The role of norB in S. aureus virulence was also tested using the silkworm Bombyx mori animal model; lethal dose 50 (LD₅₀) assays revealed slightly higher virulence in larvae infected with the wild-type strain compared to the norB mutant. Conclusions: The PC storage environment enhances quinolone resistance in S. aureus and induces differential expression of efflux pump nor genes. Furthermore, our preliminary data of the involvement of NorB in virulence of S. aureus using a silkworm model merit further investigation with other systems such as a mammal animal model. Our results provide mechanistic insights to aid clinicians in the selection of antimicrobial treatment of patients receiving transfusions of S. aureus-contaminated PCs. Full article

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

Ganglioside-induced differentiation-associated protein 2 (GDAP2) is a gene involved in hereditary cerebellar ataxia. At present, little is known about the function of GDAP2 in insects. In this study, BmGDAP2 was detected to be highly expressed in the head, epidermis, midgut, and anterior silk glands of silkworms. We generated a knockout mutant, BmGDAP2 (BmGDAP2^KO), using the CRISPR/Cas9 system. Compared with that of the wild-type, the growth cycle of BmGDAP2^KO larvae was significantly prolonged, while their body size was reduced. Furthermore, we found 149 differentially expressed genes (DEGs) between BmGDAP2^KO and the wild-type, including 106 upregulated and 43 downregulated genes. GO annotation analysis indicated that BmGDAP2 primarily influences structural and molecular activities, as well as catalytic and binding functions. KEGG pathway analysis revealed that the differentially expressed genes were mainly enriched in pathways related to peroxidase activity, hormone synthesis, apoptosis, and longevity regulation. Further investigation focused on candidate genes related to these pathways. We found that the expression levels of MAD2L1, which can inhibit cell proliferation and promote apoptosis, and Aurka-b, which plays a crucial role in cell cycle regulation, were significantly reduced in BmGDAP2^KO silkworms. These changes may interfere with the normal functions of cell division, leading to the prolonged developmental cycle observed in BmGDAP2^KO larvae. Our findings demonstrate that knockout of BmGDAP2 significantly prolongs the life cycle of Bombyx mori by affecting genes related to autophagy, apoptosis, and hormone regulation. Full article

It was found that the serine protease inhibitors BmSPI38 and BmSPI39 in silkworm can strongly inhibit the activity of porcine pancreatic elastase, which has potential applicational value in the drug research and development of lung diseases, inflammatory diseases, and skin aging caused by the excessive release of elastase. Previous studies have shown that homotypic multimers obtained by tandem expression can significantly enhance the antifungal activity and structural homogeneity of BmSPI38 and BmSPI39, while the effect of the tandem expression of these two inhibitors, with different combinations, on the total activity and expression levels of multimers remains unclear. The aim of this study is to explore whether it is possible to obtain the combination of BmSPI38 and BmSPI39 with strong total expression activity by protein engineering. In this study, 40 tandem multimer expression vectors with different combinatorial forms of BmSPI38 and BmSPI39 were constructed by the isocaudomer method, and recombinant proteins were obtained by the prokaryotic expression system. The target proteins were separated by SDS-PAGE to analyze the expression levels of multimer proteins with different combinatorial forms. The total activity of the recombinant expression products with different tandem forms was investigated using the in-gel activity staining technique of protease inhibitors. The SDS-PAGE results show that the expression levels of tandem multimers containing the BmSPI39 module at the carboxyl terminus were generally higher in the Escherichia coli supernatant than that of the tandem multimers containing the BmSPI38 module at the carboxyl terminus. The activity staining results indicate that compared with BmSPI38 and BmSPI39 homotypic multimers, the total activity of some recombinant expression products with different tandem forms was stronger. Furthermore, the total activity level was relatively higher when the carboxyl terminus of the multimer was a BmSPI39 module, such as the tandem dimers SPIAB and SPIaB and the tandem trimers SPIabB, SPIaaB, and SPIbaB. In this study, the expression of tandem fusion proteins with different combinations of the silkworm protease inhibitors BmSPI38 and BmSPI39 in E. coli was successfully achieved. It was confirmed that the tandem of different combinatorial forms, based on protein engineering, was an effective way to enhance the total activity of the fusion proteins of BmSPI38 and BmSPI39 and to improve their expression levels. Additionally, a number of multimer proteins with strong total activity and high exogenous expression levels were also screened, for example, SPIbaA, SPIbbA, SPIbbB, SPIabB, SPIaaB, and SPIbaB. This study not only lays the foundation for the exogenous production and development of BmSPI38 and BmSPI39 but also provides a reference for the construction of tandem and multimerization exploration of other protease inhibitors. Full article

Spider silk is part of a special class of natural protein fibers that have high strength and toughness: these materials have excellent comprehensive properties that are not found in other natural fibers (including silk) or most synthetic fibers. Spider egg case filaments have good hardness, can resist water, can protect spider eggs from external threats, have a significantly high initial modulus and high moisture absorption rate, and are expected to be used as a new generation of environmentally friendly natural polymer fibers and biomaterials. However, spiders are predatory and difficult to rear in large numbers, and it is also difficult to obtain spider egg case filaments in large quantities. Silkworms and spiders have a similar spinning system, and the use of transgenic technology in silkworms can obtain stable and high-yield exogenous gene proteins for a long time, representing an ideal bioreactor for the production of spider silk. In this study, the eukaryotic bioreactor and piggyBac transposon system were employed to recombinantly introduce the egg case silk protein of Nephila clavata (Nc-CYSP1) into the silkworm in the silkworm heavy-chain expression system. The results revealed that the silk glands produced a new type of transgenic silk with a significantly high initial modulus and high moisture absorption. In summary, this study provides an experimental reference for future research on the large-scale production and application of spider egg case filamentous protein, with great application prospects in the development of new environmentally friendly materials. Full article

The classic model of sex determination in insects suggests that they do not have sex hormones and that sex is determined in a cell-autonomous manner. On the other hand, there is accumulating evidence that the development of secondary sexual traits is controlled in a non-cell-autonomous manner through external factors. To evaluate the degrees of the cell-autonomous and non-cell-autonomous regulation of secondary sexual trait development, we analyzed the dynamics of the sexually dimorphic transcriptome in gynandromorphic individuals of the mo mutant strain in the silkworm Bombyx mori. The silkworm possesses a female heterogametic sex-determination system (ZZ = male/ZW = female), where the master regulatory gene for femaleness, Feminizer (Fem), is located in the W chromosome. As a secondary sexual trait, we focused on the fat body, which shows remarkable differences between the sexes during the last instar larval stage. A comparison of the transcriptomes between the fat bodies of male and female larvae identified 232 sex-differentially expressed genes (S-DEGs). The proportions of ZZ and ZW cells constituting the fat body of the gynandromorphic larvae were calculated according to the expression level of the Fem. Based on the obtained values, the expression level of each S-DEG was estimated, assuming that the levels of S-DEG expression were determined according to the proportion of ZZ and ZW cells. The estimated expression levels of 207 out of 232 S-DEGs were strongly correlated with the corresponding S-DEG expression level of the gynandromorphic fat body, determined by RNA-seq. These results strongly suggest that most of the sexually dimorphic transcriptome in the fat body is regulated in a cell-autonomous manner. Full article

The increasing prevalence of diabetic reproductive complications has prompted the development of innovative animal models. The use of the silkworm Bombyx mori as a model for diabetic reproductive damage shows potential as a valuable research tool. This study employed silkworms as a novel model to investigate diabetic reproductive damage. The silkworms were fed a high-glucose diet containing 10% glucose to induce a diabetic model. Subsequently, the study concentrated on assessing the influence of diabetes on the reproductive system of male silkworms. The results indicate that diabetes resulted in reduced luteinizing hormone (LH) and testosterone (T) levels, as well as elevated triglyceride (TG) levels in male silkworms. Moreover, diabetes mellitus was associated with pathological testicular damage in male silkworms, accompanied by decreased glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels, along with increased malondialdehyde (MDA) levels in the testis. Additionally, diabetes mellitus reduced the expression of siwi1 and siwi2 genes in the testis of male silkworms. Overall, these results support using silkworms as a valuable model for studying diabetic reproductive damage. Full article

Recent studies have suggested that ABC transporters are the main receptors of Cry toxins. However, the receptors of many Cry toxins have not been identified. In this study, we used a heterologous cell expression system to identify Bombyx mori ABC transporter subfamily C members (BmABCCs) that function as receptors for five Cry toxins active in Lepidopteran insects: Cry1Aa, Cry1Ca, Cry1Da, Cry8Ca, and Cry9Aa. All five Cry toxins can use multiple ABCCs as low-efficiency receptors, which induce cytotoxicity only at high concentrations. Surface plasmon resonance analysis revealed that the K_D values between the toxins and BmABCC1 and BmABCC4 were 10⁻⁵ to 10⁻⁹ M, suggesting binding affinities 8- to 10,000-fold lower than those between Cry1Aa and BmABCC2, which are susceptibility-determining receptors for Cry1Aa. Bioassays in BmABCC-knockout silkworm strains showed that these low-efficiency receptors are not involved in sensitivity to Cry toxins. The findings suggest that each family of Cry toxins uses multiple BmABCCs as low-efficiency receptors in the insect midgut based on the promiscuous binding of their receptor-binding regions. Each Cry toxin seems to have evolved to utilize one or several ABC transporters as susceptibility-determining receptors. Full article

The resistance of silkworms to Bombyx mori nuclear polyhedrosis virus (BmNPV) is controlled by a major dominant gene and multiple modifying genes. Given the presence of modified genes, it is difficult to determine the main gene by positional cloning. In this study, the main anti-BmNPV gene of BmNPV-resistant silkworm variety N was introduced into the susceptible variety Su to breed the near-isogenic line SuN with BmNPV resistance. The infection process of BmNPV in the hemolymph of Su and SuN was analyzed using the cell analysis system TissueFAXS PLUS. According to the law of infection and proliferation, hemolymph was extracted every 6 h for two-dimensional electrophoresis (2-DE) analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Seven DEPs were found in comparisons between Su and SuN by 2-DE analysis. Among them, acid phosphatase, storage protein, and phenoloxidase can prevent pathogen invasion, which may play a role against BmNPV. Polyamine oxidase plays an important role in energy metabolism, which may be indirectly involved in the process of resisting BmNPV. Most of the transcriptional expression profiles of the seven DEPs were consistent with the 2-DE results. This study can provide a reference for the identification of anti-BmNPV genes and the breeding of BmNPV-resistant silkworm varieties. Full article

The prophenoloxidase (PPO) activation and Toll antimicrobial peptide synthesis pathways are two critical immune responses in the insect immune system. The activation of these pathways is mediated by the cascade of serine proteases, which is negatively regulated by serpins. In this study, we identified a typical serpin, BmSerpin-4, in silkworms, whose expression was dramatically up-regulated in the fat body and hemocytes after bacterial infections. The pre-injection of recombinant BmSerpin-4 remarkably decreased the antibacterial activity of the hemolymph and the expression of the antimicrobial peptides (AMPs) gloverin-3, cecropin-D, cecropin-E, and moricin in the fat body under Micrococcus luteus and Yersinia pseudotuberculosis serotype O: 3 (YP III) infection. Meanwhile, the inhibition of systemic melanization, PO activity, and PPO activation by BmSerpin-4 was also observed. Hemolymph proteinase 1 (HP1), serine protease 2 (SP2), HP6, and SP21 were predicted as the candidate target serine proteases for BmSerpin-4 through the analysis of residues adjacent to the scissile bond and comparisons of orthologous genes in Manduca sexta. This suggests that HP1, SP2, HP6, and SP21 might be essential in the activation of the serine protease cascade in both the Toll and PPO pathways in silkworms. Our study provided a comprehensive characterization of BmSerpin-4 and clues for the further dissection of silkworm PPO and Toll activation signaling. Full article

The Duox-ROS defense system plays an important role in insect intestinal immunity. To investigate the role of intestinal microbiota in Duox-ROS regulation herein, 16S rRNA sequencing technology was utilized to compare the characteristics of bacterial populations in the midgut of silkworm after different time-periods of treatment with three feeding methods: 1–4 instars artificial diet (AD), 1–4 instars mulberry leaf (ML) and 1–3 instars artificial diet + 4 instar mulberry leaf (TM). The results revealed simple intestinal microbiota in the AD group whilst microbiota were abundant and variable in the ML and TM silkworms. By analyzing the relationship among intestinal pH, reactive oxygen species (ROS) content and microorganism composition, it was identified that an acidic intestinal environment inhibited the growth of intestinal microbiota of silkworms, observed concurrently with low ROS content and a high activity of antioxidant enzymes (SOD, TPX, CAT). Gene expression associated with the Duox-ROS defense system was detected using RT-qPCR and identified to be low in the AD group and significantly higher in the TM group of silkworms. This study provides a new reference for the future improvement of the artificial diet feeding of silkworm and a systematic indicator for the further study of the relationship between changes in the intestinal environment and intestinal microbiota balance caused by dietary alterations. Full article

Chromium is a severe heavy metal pollutant with significant environmental risks. The effects of Chromium on the digestion of Bombyx mori (silkworms) are of particular importance due to their ecological and economic significance. Herein, RNA sequencing was conducted on nine midgut samples from silkworms exposed to control, 12 g/kg and 24 g/kg Chromium chemical diets. Comparative transcriptomics revealed that under moderate Chromium exposure, there was a significant increase in up-regulated genes (1268 up-regulated to 857 down-regulated), indicating a stimulation response. At higher stress levels, a weakened survival response was observed, with a decrease in up-regulated genes and an increase in down-regulated genes (374 up-regulated to 399 down-regulated). A notable shift in cellular responses under medium chromium exposure was exposed, signifying the activation of crucial metabolic and transport systems and an elevation in cellular stress and toxicity mechanisms. The observation of up-regulated gene expression within xenobiotic metabolism pathways suggests a heightened defense against Chromium-induced oxidative stress, which was primarily through the involvement of antioxidant enzymes. Conversely, high-dose Chromium exposure down-regulates the folate biosynthesis pathway, indicating biological toxicity. Two novel genes responsive to pressure were identified, which could facilitate future stress adaptation understanding. The findings provide insights into the molecular mechanisms underlying silkworms’ digestion response to Chromium exposure and could inform its biological toxicity. Full article

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious threat to sericulture. Nevertheless, no effective control strategy is currently available. The innate immunity of silkworm is critical in the antiviral process. Exploring its molecular mechanism provides theoretical support for the prevention and treatment of BmNPV. Insect hormone receptors play an essential role in regulating host immunity. We found a correlation between Bombyx mori ecdysone receptor B1 (BmEcR-B1) and BmNPV infection, whereas the underlying mechanism remains unclear. In this study, the expression patterns and sequence characteristics of BmEcR-B1 and its isoform, BmEcR-A, were initially analyzed. BmEcR-B1 was found to be more critical than BmEcR-A in silkworm development and responses to BmNPV. Moreover, RNAi and an overexpression in BmN cells showed BmEcR-B1 had antiviral effects in the presence of 20-hydroxyecdysone (20E); Otherwise, it had no antiviral activity. Furthermore, BmEcR-B1 was required for 20E-induced apoptosis, which significantly suppressed virus infection. Finally, feeding 20E had no significant negative impacts on larval growth and the cocoon shell, suggesting the regulation of this pathway has practical value in controlling BmNPV in sericulture. The findings of this study provide important theoretical support for understanding the mechanism of the silkworm innate immune system in response to BmNPV infection. Full article

DNA N6-methyladenine (6mA) has recently been found to play regulatory roles in gene expression that links to various biological processes in eukaryotic species. The functional identification of 6mA methyltransferase will be important for understanding the underlying molecular mechanism of epigenetic 6mA methylation. It has been reported that the methyltransferase METTL4 can catalyze the methylation of 6mA; however, the function of METTL4 remains largely unknown. In this study, we aim to investigate the role of the Bombyx mori homolog METTL4 (BmMETTL4) in silkworm, a lepidopteran model insect. By using CRISPR-Cas9 system, we somatically mutated BmMETTL4 in silkworm individuates and found that disruption of BmMETTL4 caused the developmental defect of late silkworm embryo and subsequent lethality. We performed RNA-Seq and identified that there were 3192 differentially expressed genes in BmMETTL4 mutant including 1743 up-regulated and 1449 down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that genes involved in molecular structure, chitin binding, and serine hydrolase activity were significantly affected by BmMETTL4 mutation. We further found that the expression of cuticular protein genes and collagens were clearly decreased while collagenases were highly increased, which had great contributions to the abnormal embryo and decreased hatchability of silkworm. Taken together, these results demonstrated a critical role of 6mA methyltransferase BmMETTL4 in regulating embryonic development of silkworm. Full article

Mulberry (Morus alba L.) has been an economically important food crop for the domesticated silkworm, Bombyx mori, in China for more than 5000 years. However, little is known about the mechanism underlying mulberry response to environmental stress. In this study, quantitative proteomics was applied to elucidate the molecular mechanism of drought response in mulberry. A total of 604 differentially expressed proteins (DEPs) were identified via LC-MS/MS. The proteomic profiles associated with antioxidant enzymes, especially five glutathione peroxidase (GPX) isoforms, as a scavenger of reactive oxygen species (ROS), were systematically increased in the drought-stressed mulberry. This was further confirmed by gene expression and enzymatic activity. Furthermore, overexpression of the GPX isoforms led to enhancements in both antioxidant system and ROS-scavenging capacity, and greater tolerance to drought stress in transgenic plants. Taken together, these results indicated that GPX-based antioxidant enzymes play an important role in modulating mulberry response to drought stress, and higher levels of GPX can improve drought tolerance through enhancing the capacity of the antioxidant system for ROS scavenging. Full article