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1.
Braz. j. biol ; 84: e249664, 2024. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1345558

RESUMO

Abstract The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.


Resumo O impacto dos antibióticos no crescimento e na produção do casulo foi avaliado, além do isolamento e caracterização das bactérias associadas ao intestino de larvas infectadas do bicho-da-seda. A criação das larvas foi mantida nas condições recomendadas de temperatura e umidade. As larvas do bicho-da-seda com sintomas anormais foram coletadas do grupo controle e dissecadas para coleta do intestino. As bactérias foram isoladas do conteúdo intestinal por espalhamento em placas de ágar e incubadas a 37° C durante 48 horas. A identificação bacteriana e a análise filogenética foram realizadas pelo sequenciamento do gene 16S rRNA. As bactérias isoladas foram submetidas a teste de sensibilidade antimicrobiana (métodos de difusão em disco) com penicilina (10 µg / mL), tetraciclina (30 µg / mL), amoxicilina (25 µg / mL), ampicilina (10 µg / mL) e eritromicina (15 µg / mL). Todas as cepas isoladas apresentaram resultados positivos para o teste da catalase. Isolamos e identificamos cepas bacterianas (n = 06) do intestino de larvas de bicho-da-seda saudáveis e doentes. Com base na sequência do gene 16S rRNA, as bactérias isoladas mostraram estreita relação com Serratia, Bacillus e Pseudomonas spp. Notavelmente, 83,3% das cepas eram resistentes a penicilina, tetraciclina, amoxicilina, ampicilina e eritromicina, mas 16,6% mostraram suscetibilidade aos antibióticos comumente usados mencionados acima. As larvas do bicho-da-seda alimentadas com folhas tratadas com penicilina apresentaram melhora significativa no peso larval, comprimento larval e produção de casulo. Peso larval significativamente maior (6,88g), comprimento larval (5,84cm) e peso do casulo (1,33g) foram registrados para larvas alimentadas com folhas tratadas com penicilina, em comparação com outros antibióticos. Cepas bacterianas isoladas mostraram estreita relação com Serratia spp., Bacillus spp. e Pseudomonas spp.


Assuntos
Animais , Bombyx , Antibacterianos/farmacologia , Filogenia , Bactérias/genética , RNA Ribossômico 16S/genética , Testes de Sensibilidade Microbiana , Larva
2.
Talanta ; 252: 123841, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36001902

RESUMO

Herein, we demonstrate a high-accuracy H2O2 selective organic-inorganic 3D-heterointerface based on catalytically in-situ reduced Prussian-blue nanoparticles (PBNPs), Poly (3,4-ethylene dioxythiophene):poly (styrene sulfonic acid) (PEDOT:PSS) and water-soluble Silkworm protein (SWp). PBNPs were immobilized on an indium tin oxide-coated glass (ITO) electrode through the electrochemical polymerization process of PEDOT:PSS and the yielding intertwining composite was templated by the use of SWp, simultaneously. Since PSS and SWp act as poly-anionic and poly-cationic charge compensating elements, the sensing system's potential cycling and amperometric response stability have been significantly enhanced thanks to the arising physical blockage effect. Constructed sensing system showed a substantially high sensitivity (1031.7 µA mM-1 cm-2) and a low limit of detection value (LOD, 0.29 µM) between 1 and 130 µM H2O2. Eliminating the possible signal disruptions by common anion and cations in tap water, the (PEDOT:PSS:PB):SWp interface successfully selected H2O2 between the concentration of 10-40 µM with high recovery and relatively low RSDs oscillating between 94.2-110.9% and 2.9-5.1%, respectively. It is thought that the proposed heterointerface can be used in the field of sensor, biosensor and fuel cell systems run by H2O2 assay based on 3D scaffold-templated conductive polymer-PBNPs network.


Assuntos
Bombyx , Nanopartículas , Animais , Peróxido de Hidrogênio , Poliestirenos , Água
3.
Gene ; 851: 146971, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36261082

RESUMO

The main function of Sec61 complex is participating in the transport of polypeptide chains across the endoplasmic reticulum. The Sec61α subunit is the largest subunit of the Sec61 complex and shows high degree of conservation. In this study, we identified the NbSec61α and NbSec61γ genes in the microsporidian Nosema bombycis for the first time. Multiple sequence alignment showed that the sequence similarity between NbSec61α and homologous proteins of other microsporidia was greater than 48 %. NbSec61α contains a "plug" domain (amino acids 40-74) unique to the Sec61/SecY complex. Phylogenetic analysis based on NbSec61α and NbSec61γ indicated that the N. bombycis was closely related to Nosema granulosis, Nosema ceranae and Nosema apis. Indirect immunfluorescence assay showed that NbSec61α and NbSec61γ were mainly distributed in the perinuclear region of N. bombycis in different developmental phases. qRT-PCR results revealed that the expression level of NbSec61α gene increased in the early stage and reached the highest at 48 h, then decreased in the late stages. After knockdown of NbSec61α, the expression of NbSec61α, NbSec61γ and NbssrRNA genes were all significantly down-regulated. These results suggest that the NbSec61α and NbSec61γ may play an important role in the intracellular development of N. bombycis.


Assuntos
Bombyx , Nosema , Animais , Filogenia , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Nosema/genética , Nosema/metabolismo , Alinhamento de Sequência , Transporte Proteico , Bombyx/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo
4.
Chemosphere ; 311(Pt 1): 137026, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36419271

RESUMO

Silkworm (Bombyx mori L.), as an economic insect, occupies a certain position in the development of China's economy. The neonicotinoid insecticide nitenpyram is commonly used in farmland to control planthoppers and aphids. In China, mulberry orchards are often planted adjacent to fields or commercial crops, and mist drifts occur during application, which may affect the production safety of Bombyx mori. In this study, a risk assessment of nitenpyram was carried out, and the results showed that there were risks in spraying nitenpyram around the periphery and subperipheries of mulberry fields. However, few studies have reported the mechanism underlying nitenpyram's toxic effect on silkworms. Here, we validated 25 differentially expressed (DE) miRNAs in the nitenpyram treatment group of silkworms, and the significantly enriched mTOR signaling pathway, oxidative phosphorylation and FoxO signaling pathway were verified. Among them, bmo-miR-2766-5P was up-regulated by 2.122-fold, and the expression of its regulated target gene 101,741,287 was up-regulated. After the injection of bmo-miR-2766-5P inhibitor, the Log2FC value of 101,741,287 was changed from 1.26 to -2.19. Bmo-miR-3326, bmo-miR-3378-5P and bmo-miR-2761-3P were down-regulated by 2.386-fold, 1.158-fold and 2.359-fold, respectively. After injecting miRNA mimics into silkworms, the Log2FC values of the target genes 100,302,609, 101,740,730 and 101,746,319 were changed from 1.24 to -11.94, -1.12 changed to 2.84 and 1.93 changed to -0.37, respectively. In addition, nitenpyram induced oxidative damage in silkworms, and the degree of DNA damage increased with the increase of concentration and time. Meanwhile Imd was significantly up-regulated in IMD-related pathways (38.7-fold, p < 0.01). The results indicated that nitenpyram could affect the growth and development process of silkworms, and these DE-miRNAs may have an important impact on the stress response of silkworms to nitenpyram.


Assuntos
Bombyx , MicroRNAs , Animais , Bombyx/metabolismo , Neonicotinoides/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , China
5.
Proc Natl Acad Sci U S A ; 119(46): e2206025119, 2022 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-36343250

RESUMO

The Lepidoptera are an insect order of cultural, economic, and environmental importance, representing ∼10% of all described living species. Yet, for all but one of these species (silkmoth, Bombyx mori), the molecular genetics of how sexual fate is determined remains unknown. We investigated this in the diamondback moth (Plutella xylostella), a globally important, highly invasive, and economically damaging pest of cruciferous crops. Our previous work uncovered a regulator of male sex determination in P. xylostella-PxyMasc, a homolog of B. mori Masculinizer-which, although initially expressed in embryos of both sexes, is then reduced in female embryos, leading to female-specific splicing of doublesex. Here, through sequencing small RNA libraries generated from early embryos and sexed larval pools, we identified a variety of small silencing RNAs (predominantly Piwi-interacting RNAs [piRNAs]) complementary to PxyMasc, whose temporal expression correlated with the reduction in PxyMasc transcript observed previously in females. Analysis of these small RNAs showed that they are expressed from tandemly arranged, multicopy arrays found exclusively on the W (female-specific) chromosome, which we term "Pxyfem". Analysis of the Pxyfem sequences showed that they are partial complementary DNAs (cDNAs) of PxyMasc messenger RNA (mRNA) transcripts, likely integrated into transposable element graveyards by the noncanonical action of retrotransposons (retrocopies), and that their apparent similarity to B. mori feminizer more probably represents convergent evolution. Our study helps elucidate the sex determination cascade in this globally important pest and highlights the "shortcuts" that retrotransposition events can facilitate in the evolution of complex molecular cascades, including sex determination.


Assuntos
Bombyx , Mariposas , Feminino , Masculino , Animais , Bombyx/genética , Bombyx/metabolismo , Mariposas/genética , Mariposas/metabolismo , Splicing de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo
6.
PLoS Pathog ; 18(11): e1010938, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36383572

RESUMO

Baculoviruses are virulent pathogens that infect a wide range of insects. They initiate infections via specific interactions between the structural proteins on the envelopes of occlusion-derived virions (ODVs) and the midgut cell surface receptors in hosts. However, host factors that are hijacked by baculoviruses for efficient infection remain largely unknown. In this study, we identified a membrane-associated protein sucrose hydrolase (BmSUH) as an ODV binding factor during Bombyx mori nucleopolyhedrovirus (BmNPV) primary infection. BmSUH was specifically expressed in the midgut microvilli where the ODV-midgut fusion happened. Knockout of BmSUH by CRISPR/Cas9 resulted in a significantly higher survival rate after BmNPV orally infection. Liquid chromatography-tandem mass spectrometry analysis and co-immunoprecipitation analysis demonstrated that PIF protein complex required for ODV binding could interact with BmSUH. Furthermore, fluorescence dequenching assay showed that the amount of ODV binding and fusion to the midgut decreased in BmSUH mutants compared to wild-type silkworm, suggesting the role of BmSUH as an ODV binding factor that mediates the ODV entry process. Based on a multilevel survey, the data showed that BmSUH acted as a host factor that facilitates BmNPV oral infection. More generally, this study indicated that disrupting essential protein-protein interactions required for baculovirus efficient entry may be broadly applicable to against viral infection.


Assuntos
Bombyx , Nucleopoliedrovírus , Animais , Proteínas de Membrana/metabolismo , Nucleopoliedrovírus/metabolismo , Sistema Digestório , Baculoviridae
7.
J Agric Food Chem ; 70(45): 14427-14438, 2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36321811

RESUMO

The Bombyx mori nucleopolyhedrovirus (BmNPV), a foodborne infectious virus, is the pathogen causing nuclear polyhedrosis and high lethality in the silkworm. In this study, we characterized the molecules involved in BmNPV-silkworm interaction by RNA sequencing of the fat body isolated from the virus-susceptible strain P50. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation showed that the upregulated differentially expressed genes (DEGs) were mainly involved in translation, signal transduction, folding, sorting, and degradation, as well as transport and catabolism, while the downregulated DEGs were predominantly enriched in the metabolism of carbohydrates, amino acids, and lipids at 72 h post BmNPV infection. Knockout of the upregulated somatomedin-B and thrombospondin type-1 domain-containing protein, probable allantoicase, trifunctional purine biosynthetic protein adenosine-3, and Psl and pyoverdine operon regulator inhibited the proliferation of BmNPV, while knockout of the downregulated clip domain serine protease 3 and carboxylesterase clade H, member 1 promoted it. The molecules herein identified provide a foundation for developing strategies and designing drugs against BmNPV.


Assuntos
Bombyx , Nucleopoliedrovírus , Animais , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/metabolismo , Bombyx/genética , Bombyx/metabolismo , Anotação de Sequência Molecular , Proliferação de Células
8.
Int J Mol Sci ; 23(21)2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36362309

RESUMO

As a consequence of long-term coevolution and natural selection, the leaves of mulberry (Morus alba) trees have become the best food source for silkworms (Bombyx mori). Nevertheless, the molecular and genomic basis of defense response remains largely unexplored. In the present study, we assessed changes in the transcriptome changes of mulberry in response to silkworm larval feeding at 0, 3, and 6 h. A total of 4709 (up = 2971, down = 1738) and 3009 (up = 1868, down = 1141) unigenes were identified after 3 and 6 h of silkworm infestation, respectively. MapMan enrichment analysis results show structural traits such as leaf surface wax, cell wall thickness and lignification form the first physical barrier to feeding by the silkworms. Cluster analysis revealed six unique temporal patterns of transcriptome changes. We predicted that mulberry promoted rapid changes in signaling and other regulatory processes to deal with mechanical damage, photosynthesis impairment, and other injury caused by herbivores within 3-6 h. LRR-RK coding genes (THE1, FER) was predicted participated in perception of cell wall perturbation in mulberry responding to silkworm feeding. Ca2+ signal sensors (CMLs), ROS (OST1, SOS3), RBOHD/F, CDPKs, and ABA were part of the regulatory network after silkworm feeding. Jasmonic acid (JA) signal transduction was predicted to act in silkworm feeding response, 10 JA signaling genes (such as OPR3, JAR1, and JAZ1) and 21 JA synthesis genes (such as LOX2, AOS, and ACX1) were upregulated after silkworm feeding for 3 h. Besides, genes of "alpha-Linolenic acid metabolism" and "phenylpropanoid biosynthesis" were activated in 3 h to reprogram secondary metabolism. Collectively, these findings provided valuable insights into silkworm herbivory-induced regulatory and metabolic processes in mulberry, which might help improve the coevolution of silkworm and mulberry.


Assuntos
Bombyx , Morus , Animais , Morus/química , Bombyx/metabolismo , Transcriptoma , Folhas de Planta/metabolismo , Perfilação da Expressão Gênica
9.
Sci Rep ; 12(1): 18502, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36323753

RESUMO

The silkworm, Bombyx mori, is an attractive host for recombinant protein production due to its high expression efficiency, quality, and quantity. Two expression systems have been widely used for recombinant protein production in B. mori: baculovirus/silkworm expression system and transgenic silkworm expression system. Both expression systems enable high protein production, but the qualities of the resulting recombinant proteins have not been well evaluated. In this study, we expressed bovine interferon γ (IFN-γ) using the two systems and examined the quality of the resulting proteins in terms of N-glycosylation and protein cleavage. Both expression systems successfully produced IFN-γ as an N-glycoprotein. Although the production in the baculovirus/silkworm expression system was much more efficient than that in the transgenic silkworm expression system, unexpected variants of IFN-γ were also produced in the former system due to the different N-glycosylation and C-terminal truncations. These results indicate that while high protein production could be achieved in the baculovirus/silkworm expression system, unintentional protein modification might occur, and therefore protein expression in the transgenic silkworm expression system is preferable from the point-of-view of N-glycosylation of the recombinant protein and evasion of unexpected attack by a protease in B. mori.


Assuntos
Bombyx , Animais , Bovinos , Bombyx/genética , Bombyx/metabolismo , Proteínas Recombinantes/metabolismo , Animais Geneticamente Modificados , Glicosilação
10.
Ecotoxicol Environ Saf ; 247: 114245, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36327780

RESUMO

Triphenyltin (TPT) is a widely used reagent in various industries and agriculture, but is also known to accumulate in natural ecosystems and animal tissues. Hence, the aim of this study was to comprehensively assess the toxicity of TPT in the silkworm Bombyx mori as a model insect. The results showed that TPT exposure for the entire 5th instar larval stage significantly reduced the weight of silkworm pupa and inhibited development of the silkworm midgut. Following exposure to 2 µg/kg of TPT for 4 days, differentially expressed genes in midgut were associated with enriched pathways involved in the metabolism of carbohydrates, lipids, and amino acids, as determined by RNA sequencing. Furthermore, the metabolic profiles of the intestinal content of silkworms exposed to 2 µg/kg of TPT for 4 days were markedly altered and differential metabolites produced by metabolism of carbohydrates, lipids, and amino acids were enriched as determined by non-targeted GC-MS/MS metabolomics. This study provides novel insights into the mechanisms underlying the toxicity of TPT and emphasizes the risks posed by such pollutants released into the environment.


Assuntos
Bombyx , Animais , Bombyx/genética , Ecossistema , Espectrometria de Massas em Tandem , Insetos , Aminoácidos , Lipídeos
11.
Viruses ; 14(11)2022 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-36366503

RESUMO

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens restricting the sustainable development of the sericulture industry. Currently, there is no effective treatment for BmNPV infection in silkworms, and the mechanism underlying BmNPV resistance in silkworms is also not clear. In this study, comparative transcriptome analyses were carried out in midguts of two silkworm varieties, namely BaiyuN, which is a resistance variety, and Baiyu, which is a susceptible variety, at five different time points (i.e., 0, 1, 3, 6, and 9 h) post-BmNPV infection to detect the early-stage transcriptional changes in these silkworms. In total, 1911 and 1577 differentially expressed genes (DEGs) were identified in the Baiyu and BaiyuN varieties, respectively, involving a total of 48 metabolic pathways. Of these pathways, eight were shared by the Baiyu and BaiyuN varieties in response to BmNPV infection. Notably, four genes (i.e., BGIBMGA08815, BGIBMGA003935, BGIBMGA003571, BGIBMGA010059) were upregulated in the Baiyu variety while downregulated in the BaiyuN variety. The inhibited expression of these four genes in the resistant variety highlighted their potential roles in the resistance of early-stage viral replication. Thus, our study provided a new avenue for the further study of the mechanism underlying BmNPV infection in silkworms and the potential treatment of BmNPV infection.


Assuntos
Bombyx , Nucleopoliedrovírus , Animais , Nucleopoliedrovírus/genética , Replicação Viral , Perfilação da Expressão Gênica
12.
Int J Mol Sci ; 23(22)2022 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-36430901

RESUMO

Silk fibroin (SF), an organic material obtained from the cocoons of a silkworm Bombyx mori, is used in several applications and has a proven track record in biomedicine owing to its superior compatibility with the human body, superb mechanical characteristics, and its controllable propensity to decay. Due to its robust biocompatibility, less immunogenic, non-toxic, non-carcinogenic, and biodegradable properties, it has been widely used in biological and biomedical fields, including wound healing. The key strategies for building diverse SF-based drug delivery systems are discussed in this review, as well as the most recent ways for developing functionalized SF for controlled or redirected medicines, gene therapy, and wound healing. Understanding the features of SF and the various ways to manipulate its physicochemical and mechanical properties enables the development of more effective drug delivery devices. Drugs are encapsulated in SF-based drug delivery systems to extend their shelf life and control their release, allowing them to travel further across the bloodstream and thus extend their range of operation. Furthermore, due to their tunable properties, SF-based drug delivery systems open up new possibilities for drug delivery, gene therapy, and wound healing.


Assuntos
Bombyx , Fibroínas , Animais , Humanos , Fibroínas/química , Materiais Biocompatíveis/farmacologia , Cicatrização , Sistemas de Liberação de Medicamentos , Bombyx/genética , Bombyx/química , Terapia Genética , Preparações Farmacêuticas
13.
Molecules ; 27(22)2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36431809

RESUMO

Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin produced by the entomopathogenic fungus Metarhizium anisopliae, has insecticidal activity, but its molecular mechanism of action is still not clear. Three proteins with modification-related functions, calreticulin (BmCRT), dipeptidyl peptidase Ⅲ (BmDPP3), and protein disulfide isomerase A5 (BmPDIA5), were selected to verify the interactions with DA in this study. The kinetic data of the interactions were measured by surface plasmon resonance (SPR) and bio-layer interferometry (BLI) in vitro. The KD values of DA with BmCRT, BmDPP3, and BmPDIA5 ranged from 10-4 to 10-5 mol/L, which suggested that the three proteins all had fairly strong interactions with DA. Then, it was found that DA in a dose-dependent manner affected the interactions of the three proteins with their partners in insect two-hybrid tests in SF-9 cells. Furthermore, the results of enzyme activities by ELISA indicated that DA could inhibit the activity of BmDPP3 but had no significant effect on BmPDIA5. In addition, DA induced the upregulation of BmDPP3 and the downregulation of BmCRT. The results prove that BmCRT, BmDPP3, and BmPDIA5 are all binding proteins of DA. This study might provide new insights to elucidate the molecular mechanism of DA.


Assuntos
Bombyx , Depsipeptídeos , Animais , Ressonância de Plasmônio de Superfície , Regulação para Baixo
14.
Nat Commun ; 13(1): 6764, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376299

RESUMO

Bacterial symbionts, such as Wolbachia species, can manipulate the sexual development and reproduction of their insect hosts. For example, Wolbachia infection induces male-specific death in the Asian corn borer Ostrinia furnacalis by targeting the host factor Masculinizer (Masc), an essential protein for masculinization and dosage compensation in lepidopteran insects. Here we identify a Wolbachia protein, designated Oscar, which interacts with Masc via its ankyrin repeats. Embryonic expression of Oscar inhibits Masc-induced masculinization and leads to male killing in two lepidopteran insects, O. furnacalis and the silkworm Bombyx mori. Our study identifies a mechanism by which Wolbachia induce male killing of host progeny.


Assuntos
Bombyx , Mariposas , Wolbachia , Masculino , Animais , Wolbachia/metabolismo , Bombyx/genética , Bombyx/metabolismo , Mariposas/microbiologia , Compensação de Dosagem (Genética) , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo
15.
Front Immunol ; 13: 1014985, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36389836

RESUMO

Poria cocos polysaccharides (PS) have been used as Chinese traditional medicine with various pharmacological effects, including antiviral, anti-oxidative, and immunomodulatory activities. Herein Bombyx mori silkworm was used as a model animal to evaluate the immunomodulatory effects of PS via detecting the changes of innate immune parameters and explore the underlying molecular mechanism of the immunoregulatory effect of PS using Illumina HiSeq Xten platform. The results presented here demonstrated that a hemocoel injection of PS significantly enhanced the cellular immunity of silkworm, including hemocyte phagocytosis, microaggregation, and spreading ability. A total of 335 differentially expressed genes (DEGs) were screened, including 214 upregulated genes and 121 downregulated genes by differential expression analysis. Gene annotation and enrichment analyses showed that many DEGs related to immune signal recognition, detoxification, proPO activation, carbohydrate metabolism, and lipid metabolism were significantly upregulated in the treatment group. The Kyoto Encyclopedia of Genes and Genomes-based Gene Set Enrichment Analysis also revealed that the more highly expressed gene sets in the PS treatment silkworm were mainly related to immune signal transduction pathways and energy metabolism. In addition, the activity of four enzymes related to immunity and energy metabolism-including phenoloxidase, glucose-6-phosphate dehydrogenase, hexokinase, and fatty acid synthetase-were all significantly increased in the larvae injected with PS. We performed qRT-PCR to examine the expression profile of immune and metabolic-related genes, which further verified the reliability of our transcriptome data and suggested that PS can regulate the immunity of silkworm by enhancing the cellular immunity and modulating the expression levels of genes related to immune responses and physiological metabolism. These findings will lay a scientific foundation for the use of PS as an immunomodulator in disease prevention in human beings or animals.


Assuntos
Bombyx , Wolfiporia , Animais , Humanos , Bombyx/genética , Bombyx/metabolismo , Wolfiporia/genética , Reprodutibilidade dos Testes , Perfilação da Expressão Gênica/métodos , Larva/genética , Polissacarídeos/farmacologia , Polissacarídeos/metabolismo
16.
Nat Commun ; 13(1): 6295, 2022 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-36273007

RESUMO

Bombyx mori silk is a super-long natural protein fiber with a unique structure and excellent performance. Innovative silk structures with high performance are in great demand, thus resulting in an industrial bottleneck. Herein, the outer layer sericin SER3 is ectopically expressed in the posterior silk gland (PSG) in silkworms via a piggyBac-mediated transgenic approach, then secreted into the inner fibroin layer, thus generating a fiber with sericin microsomes dispersed in fibroin fibrils. The water-soluble SER3 protein secreted by PSG causes P25's detachment from the fibroin unit of the Fib-H/Fib-L/P25 polymer, and accumulation between the fibroin layer and the sericin layer. Consequently, the water solubility and stability of the fibroin-colloid in the silk glandular cavity, and the crystallinity increase, and the mechanical properties of cocoon fibers, moisture absorption and moisture liberation of the silk also improve. Meanwhile, the mutant overcomes the problems of low survival and abnormal silk gland development, thus enabling higher production efficiency of cocoon silk. In summary, we describe a silk gland transgenic target protein selection strategy to alter the silk fiber structure and to innovate its properties. This work provides an efficient and green method to produce silk fibers with new functions.


Assuntos
Bombyx , Fibroínas , Sericinas , Animais , Bombyx/genética , Bombyx/metabolismo , Seda/química , Sericinas/genética , Sericinas/metabolismo , Fibroínas/metabolismo , Expressão Ectópica do Gene , Animais Geneticamente Modificados , Água/metabolismo
17.
Protein J ; 41(6): 596-612, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36282463

RESUMO

SWPs are the major virulence component of microsporidian spores. In microsporidia, SWPs can be found either in exospore or endospore to serve as a putative virulence factor for host cell invasion. SWP5 is a vital protein that involves in exospore localization and supports the structural integrity of the spore wall and this action potentially modulates the course of infection in N. bombycis. Here we report recombinant SWP5 purification using Ni-NTA IMAC and SEC. GFC analysis reveals SWP5 to be a monomer which correlates with the predicted theoretical weight and overlaps with ovalbumin peak in the chromatogram. The raised polyclonal anti-SWP5 antibodies was confirmed using blotting and enterokinase cleavage experiments. The resultant fusion SWP5 and SWP5 in infected silkworm samples positively reacts to anti-SWP5 antibodies is shown in ELISA. Immunoassays and Bioinformatic analysis reveal SWP5 is found to be localized on exospore and this action could indicate the probable role of SWP5 in host pathogen interactions during spore germination and its contribution to microsporidian pathogenesis. This study will support development of a field-based diagnostic kit for the detection N. bombycis NIK-1S infecting silkworms. The analysis will also be useful for the formulation of drugs against microsporidia and pebrine disease.


Assuntos
Bombyx , Nosema , Animais , Esporos Fúngicos/genética , Esporos Fúngicos/química , Esporos Fúngicos/metabolismo , Proteínas Fúngicas/química , Nosema/genética , Nosema/química , Nosema/metabolismo , Bombyx/genética , Clonagem Molecular
18.
Proc Biol Sci ; 289(1985): 20221427, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36285499

RESUMO

Gustatory systems in phytophagous insects are used to perceive feeding stimulants and deterrents, and are involved in insect decisions to feed on particular plants. During the process, gustatory receptors (Grs) can recognize diverse phytochemicals and provide a molecular basis for taste perception. The silkworm, as a representative Lepidoptera species, has developed a strong feeding preference for mulberry leaves. The mulberry-derived flavonoid glycoside, isoquercetin, is required to induce feeding behaviours. However, the corresponding Grs for isoquercetin and underlying molecular mechanisms remain unclear. In this study, we used molecular methods, voltage clamp recordings and feeding assays to identify silkworm BmGr63, which was tuned to isoquercetin. The use of qRT-PCR confirmed that BmGr63 was highly expressed in the mouthpart of fourth and fifth instar larvae. Functional analysis showed that oocytes expressing BmGr63 from the 'bitter' clade responded to mulberry extracts. Among 20 test chemicals, BmGr63 specifically recognized isoquercetin. The preference for isoquercetin was not observed in BmGr63 knock-down groups. The tuning between BmGr63 and isoquercetin has been demonstrated, which is meaningful to explain the silkworm-mulberry feeding mechanism from molecular levels and thus provides evidence for further feeding relationship studies between phytophagous insects and host plants.


Assuntos
Bombyx , Proteínas de Drosophila , Morus , Animais , Bombyx/fisiologia , Paladar , Receptores de Superfície Celular , Insetos , Plantas , Flavonoides , Glicosídeos
19.
Insect Biochem Mol Biol ; 150: 103849, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36209956

RESUMO

Phosphomevalonate kinase (PMK) is an important enzyme involved in the juvenile hormone (JH) biosynthesis pathway that catalyzes the phosphorylation of mevalonate 5-phosphate into mevalonate 5-diphosphate in the mevalonate pathway. Herein, we report the crystal structure of insect PMK from Bombyx mori (BmPMK) at a resolution of 1.60 Å. The overall structure of BmPMK adopts a compact α/ß conformation with two parts: the core and lid regions. The interface between the core and lid regions forms a continuous and negatively charged groove to accommodate the substrates. Using computational simulation combined with site-directed mutagenesis and biochemical analysis, we define the binding mode of BmPMK with the cofactor and the substrate, which provides a structural basis for understanding the catalytic mechanism and the design of inhibitors of PMK. Moreover, BmPMK showed the optimal enzyme activity at pH 8.0, and the optimal temperature was 30 °C, using mevalonate 5-phosphate as the substrate. The expression profiles and kinetic analyses of BmPMK indicated that it plays critical role in the control of JH biosynthesis in silkworms. Collectively, these findings provide a better understanding of the structural and biochemical features of insect PMK.


Assuntos
Bombyx , Animais , Bombyx/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Mutagênese Sítio-Dirigida
20.
Int J Mol Sci ; 23(20)2022 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-36292934

RESUMO

Tetrahydrobiopterin (BH4) is a vital coenzyme for several enzymes involved in diverse enzymatic reactions in animals, and BH4 deficiency can lead to metabolic and neurological disorders due to dysfunction in its metabolism. In the silkworm natural homozygous mutant leml, the key enzyme sepiapterin reductase (BmSPR) in the de novo synthesis pathway of BH4 is inactivated, resulting in severe deficiency of BH4 synthesis. However, it is not known why the leml larvae can survive to the second-instar stage and which pathways lead to their death when BH4 is deficient. Here, we quantified BH4 and found that the fertilized eggs contained large amounts of BH4 transferred from the mother to the offspring, maintaining its normal development in the embryo and the first instar. Subsequently, we investigated the multiple pathways in which BH4 is involved as a cofactor. The results showed that BH4 deficiency in silkworms blocked the melanin synthesis pathway, caused an insufficient degree of epidermal sclerosis, disordered tyrosine metabolism, and damaged mitochondria. On the other hand, BH4 deficiency led to the uncoupling of nitric oxide synthase (BmNOS), a reduced NO production, and a significantly reduced fat in fat body catalyzation by phospholipase A2, resulting in an impaired immune system. Meanwhile, the uncoupling of BmNOS increased the O2- content, damaged the DNA, and caused the apoptosis of the body cells. Taken together, BH4 is critical for the life and death of leml mutants. This study lays a foundation for the further exploration of lepidopteran insects and provides an important basis for the treatment of human BH4 deficiency-related diseases.


Assuntos
Bombyx , Fenilcetonúrias , Animais , Humanos , Bombyx/metabolismo , Melaninas/metabolismo , Biopterina/metabolismo , Fenilcetonúrias/metabolismo , Óxido Nítrico Sintase/metabolismo
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