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1.
Pestic Biochem Physiol ; 174: 104824, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33838717

RESUMO

Acetamiprid is a new type of nicotinic insecticide that is widely used in pest control. Its environmental residues may cause silkworm cocooning disorder. In this study, silkworms that received continuous feeding of low concentration acetamiprid (0.15 mg/L) showed significantly decreased silk gland index and cocooning rate. Gene expression profiling of posterior silk glands (PSGs) revealed that the differentially expressed genes were significantly enriched in oxidative stress-related signal pathways with significant up-regulation. The contents of both H2O2 and MDA were increased, along with significantly elevated SOD and CAT activities, all of which reached maximal values at 48 h when H2O2 and MDA's contents were 10.46 and 7.98 nmol/mgprot, respectively, and SOD and CAT activities were 5.51 U/mgprot and 33.48 U/gprot, respectively. The transcription levels of antioxidant enzyme-related genes SOD, Mn-SOD, CuZn-SOD, CAT, TPX and GPX were all up-regulated, indicating that exposure to low concentration acetamiprid led to antioxidant response in silkworm PSG. The key genes in the FoxO/CncC/Keap1 signaling pathway that regulates antioxidant enzyme activity, FoxO, CncC, Keap1, NQO1, HO-1 and sMaf were all up-regulated during the whole process of treatment, with maximal values being reached at 72 h with 2.91, 1.46, 1.82, 2.52, 2.32 and 4.01 times of increases, respectively. These results demonstrate that exposure to low concentration acetamiprid causes oxidative stress in silkworm PSG, which may be the cause of cocooning disorder in silkworm. Our study provides a reference for the safety evaluation of environmental residues of acetamiprid on non-target insects.


Assuntos
Bombyx , Animais , Bombyx/genética , Bombyx/metabolismo , Crescimento e Desenvolvimento , Peróxido de Hidrogênio , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2/metabolismo , Neonicotinoides , Estresse Oxidativo , Seda
2.
Int J Mol Sci ; 22(5)2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800168

RESUMO

Transgenic silkworm expression systems have been applied for producing various recombinant proteins. Knocking out or downregulating an endogenous silk protein is considered a viable strategy for improving the ability of transgenic expression systems to produce exogenous proteins. Here, we report the expression of human epidermal growth factor (hEGF) in a P25 gene knockout silkworm. The hEGF gene regulated by the P25 gene promoter was integrated into a silkworm's genome. Five transgenic positive silkworm lineages were generated with different insertion sites on silkworm chromosomes and the ability to synthesize and secrete proteins into cocoons. Then, a cross-strategy was used to produce transgenic silkworms with a P25 gene knockout background. The results of the protein analysis showed that the loss of an endogenous P25 protein can increase the hEGF production to about 2.2-fold more than normal silkworms. Compared to those of transgenic silkworms with wild type (non-knockout) background, the morphology and secondary structure of cocoon silks were barely changed in transgenic silkworms with a P25 gene knockout background, indicating their similar physical properties of cocoon silks. In conclusion, P25 gene knockout silkworms may become an efficient bioreactor for the production of exogenous proteins and a promising tool for producing various protein-containing silk biomaterials.


Assuntos
Animais Geneticamente Modificados , Bombyx , Fator de Crescimento Epidérmico , Fibroínas/genética , Técnicas de Silenciamento de Genes , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bombyx/genética , Bombyx/metabolismo , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Arch Insect Biochem Physiol ; 106(4): e21783, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33719082

RESUMO

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


Assuntos
Ácido Ascórbico/metabolismo , Bombyx/metabolismo , Animais , Bombyx/crescimento & desenvolvimento , Hexoses/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Açúcares Ácidos/metabolismo
4.
Arch Insect Biochem Physiol ; 106(3): 1-12, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33619747

RESUMO

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


Assuntos
Bombyx , RNA Longo não Codificante , Viroses , Animais , Bombyx/genética , Bombyx/imunologia , Bombyx/metabolismo , Bombyx/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes de Insetos , Interações entre Hospedeiro e Microrganismos , Imunidade , Larva/genética , Larva/imunologia , Larva/metabolismo , RNA Longo não Codificante/isolamento & purificação , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Reoviridae , Viroses/imunologia , Viroses/metabolismo
5.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443213

RESUMO

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkworm Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.


Assuntos
Bombyx/metabolismo , Diapausa/genética , Desenvolvimento Embrionário/genética , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Proteínas de Insetos/metabolismo , Neuropeptídeos/metabolismo , Ácido gama-Aminobutírico/metabolismo , Sequência de Aminoácidos , Animais , Bombyx/embriologia , Bombyx/genética , Membrana Celular/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Antagonistas de Receptores de GABA-A/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Proteínas de Insetos/genética , Neuropeptídeos/genética , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Filogenia , Receptores de GABA-A/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
6.
Arch Insect Biochem Physiol ; 106(2): e21762, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33415772

RESUMO

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


Assuntos
Bombyx/genética , Bombyx/virologia , Genes de Insetos , Interações Hospedeiro-Patógeno , Nucleopoliedrovírus/fisiologia , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Corpo Adiposo/metabolismo , Trato Gastrointestinal/metabolismo , Hemolinfa/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/virologia
7.
Int J Biol Macromol ; 169: 473-479, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33358779

RESUMO

Fibroin of the silkworm consists of fibroin heavy chain (Fib-H) with hydrophobic intermediate repeats flanked by hydrophilic N and C terminal domains (NTD and CTD, respectively), fibroin light chain (Fib-L), and P25. However, the respective roles of each polypeptide in silk processing remain largely unknown. Here, a series of transgenic silkworms with different fusion gene expression cassettes were created in order to selectively express different fluorescent fusion proteins in silk glands. The roles of different components in silk processing were investigated via observing and analyzing the movement and distribution of these proteins in the silk gland and in cocoon silk. The data showed that hydrophilic NTDs were distributed on the surface of micelles, providing sufficient electrostatic repulsion to prevent premature crystallization of silk proteins. Hydrophilic CTD==Ls ("==" represents the disulfide bond) were located on the inner layer of micelles to control the solubility of large micelles. The results presented here elucidated the underlying mechanisms of silkworm silk processing in vivo. This is significant for the development of artificial spinning technology, novel silk biomaterials, and silk gland expression systems.


Assuntos
Bombyx/metabolismo , Fibroínas/química , Fibroínas/metabolismo , Animais , Animais Geneticamente Modificados/genética , Materiais Biocompatíveis/metabolismo , Secreções Corporais/metabolismo , Bombyx/química , Fibroínas/fisiologia , Proteínas de Insetos/genética , Domínios Proteicos/fisiologia , Seda/metabolismo
8.
Carbohydr Polym ; 253: 117214, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33278979

RESUMO

Nanofibrils derived from natural biopolymers have received extensive interest due to their exceptional mechanical properties and excellent biocompatibility. To fabricate biocompatible chitosan nanocomposites with high mechanical performance, silkworm silks were deconstructed into nanofibrils as structural and mechanical reinforcement of chitosan. After dispersing silk nanofibrils in chitosan solution, a set of nanocomposites, including film, porous scaffold, filament, and nanofibrous sponge, could be fabricated from the blended solutions. Silk nanofibrils could be uniformly dispersed in chitosan solution, and formed multi-dimensional nanocomposites. The nanocomposites exhibited enhanced mechanical strength and thermal stability, and provided a biomimetic nanofibrous structure for biomaterial applications. The enhancement in mechanical properties can be attributed to the interaction between the nanofibril phase and the chitosan matrix. As the polysaccharide/protein bionanocomposites derived from natural biopolymers, these materials offer new opportunities for biomaterial application by virtue of their biocompatibility and biodegradability, as well as enhanced mechanical properties and controllable mesoscopic structure.


Assuntos
Materiais Biocompatíveis/química , Bombyx/metabolismo , Quitosana/química , Nanocompostos/química , Nanofibras/química , Seda/química , Tecidos Suporte/química , Animais , Plásticos Biodegradáveis/química , Porosidade , Temperatura , Resistência à Tração , Engenharia Tecidual/métodos
9.
Chemosphere ; 262: 127891, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32799150

RESUMO

Fluoride generally exists in the natural environment, and has been reported to induce serious environmental hazard to animals, plants, and even humans via ecological cycle. Silkworm, Bombyx mori, which showed significant growth and reproductivity reduction when exposed to fluoride, has become a model to evaluate the toxicity of fluoride. However, the detailed mechanism underlying fluoride toxicity and corresponding transport proteins remain unclear. In this study, we performed RNA-seq of the larval midgut and fat body with fluoride exposure and normal treatment. Differential analysis showed that there were 4405 differentially expressed genes in fat body and 4430 DEGs in midgut with fluoride stress. By Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, we identified several key pathways involved in the fluoride exposure and poisoning. We focused on the oxidative phosphorylation and MAPK signal pathway. QRT-PCR confirmed that oxidative phosphorylation process was remarkably inhibited by fluoride exposure and resulted in the blocking of ATP synthesis. The MAPK signal pathway was stimulated via phosphorylation signal transduction. Moreover, by protein structure analysis combined with the DEGs, we screen 36 potential membrane proteins which might take part in transporting fluoride. Taken together, the results of our study expanded the underlying mechanisms of fluoride poisoning on silkworm larval growth and development, and implied potential fluoride transport proteins in silkworm.


Assuntos
Bombyx/fisiologia , Fluoretos/toxicidade , Substâncias Perigosas/toxicidade , Tecido Adiposo/metabolismo , Animais , Bombyx/metabolismo , Sistema Digestório/metabolismo , Corpo Adiposo/metabolismo , Perfilação da Expressão Gênica/métodos , Inativação Metabólica , Larva/genética , Transcriptoma/fisiologia
10.
J Ethnopharmacol ; 266: 113448, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33022342

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Silkworm faeces are the dry faeces of the insect Bombyx mori (Linnaeus) and have historically been used in traditional Chinese medicine to treat blood deficiency and rheumatic pain. Silkworm faeces extract (SFE) is derived from silkworm faeces. AIM OF THE STUDY: Clinical observations of patients in the Department of Nephrology have shown that SFE effectively improves renal anaemia. However, the molecular mechanism remains unclear. This article mainly explores the regulatory effects of SFE on erythropoietin (EPO) and hepcidin to identify the molecular mechanism of SFE. MATERIALS AND METHODS: A rat model of renal anaemia was established by feeding rats food containing 0.75% adenine. SFE was orally administered to the rats, while recombinant human erythropoietin (rhEPO) was used as a positive control drug. Haematological parameters and inflammation levels were compared between rats from each group, and pathological kidney sections from each rat were observed. The serum EPO and hepcidin levels were detected using enzyme-linked immunosorbent assay (ELISA) kits, while Western blot analyses were performed to detect the levels of proteins involved in the EPO-related hypoxia-inducible factor 2α (HIF-2α)/prolyl hydroxylase 2 (PHD2) signalling pathway and hepcidin-related BMP6/SMAD4 and interleukin-6 (IL-6)/STAT3 signalling pathways. RESULTS: SFE significantly ameliorated haematological parameters, renal function, and inflammation levels in the rats. A mechanistic study showed that SFE promoted EPO expression by upregulating HIF-2α expression and inhibiting the expression of NF-κB and GATA2 both in vivo and in vitro. In particular, SFE inhibited PHD2 expression, resulting in a decrease in the enzymatic reaction of HIF-2α to increase EPO expression. Furthermore, SFE inhibited hepcidin expression by blocking the BMP6/SMAD4 and IL-6/STAT3 pathways. CONCLUSIONS: SFE regulated iron metabolism by inhibiting hepcidin and simultaneously promoted EPO synthesis to improve renal anaemia in rats.


Assuntos
Anemia/prevenção & controle , Bombyx/metabolismo , Fezes/química , Nefropatias/complicações , Adenina , Anemia/etiologia , Animais , Modelos Animais de Doenças , Eritropoetina/administração & dosagem , Eritropoetina/metabolismo , Hepcidinas/antagonistas & inibidores , Hepcidinas/metabolismo , Humanos , Ferro/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley
11.
Ecotoxicol Environ Saf ; 209: 111759, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33341695

RESUMO

Ionic liquids (ILs) have been reported to be a potential water and soil pollutant, whose toxicity has gained much attention in recent years. In this work, silkworm larvae were used as a novel in vivo model to assess the biotoxicity of ILs, which were performed by three steps. The first step was to determine the susceptibility of different silkworm strains to ILs. Data showed that Jingsong×haoyue was the most susceptible one among three silkworm strains (Jingsong×haoyue, P50, and Yi16) for evaluating the biological effects of ILs. The second step was to compare the toxicity of ILs with different structures using the larvae of Jingsong×haoyue. It was found that three representative ILs, 1-octyl-3-methylimidazole chloride ([C8mim]Cl), N-octyl-3-methylpyridine chloride ([C8mpy]Cl), and 1-octyl-3-methylimidazole tetrafluoroborate ([C8mim]BF4), had significant toxic effects on the growth and development of the larvae with 24 h median lethal concentration (24 h-LC50) values of 112.3, 156.3, and 68.9 µg g-1, respectively, indicating that the types of anions and cations had impacts on the toxicity of ILs. The last step was targeted at investigating responses of the larvae to the exposure of ILs. It was observed that remarkable physiological and biochemical responses occurred in different tissues of the larvae. For example, activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) in different tissues increased significantly to form an active protective mechanism for alleviating the toxic effects of ILs. Additionally, an increase of malondialdehyde (MDA) contents was found in the larvae. The data suggested that ILs could induce lipid peroxidation and cellular damage, which may be the main reason for toxicity of ILs to the larvae. Therefore, silkworm larvae could be used as a susceptible and reliable in vivo model to evaluate the toxicity of ILs, and the results are helpful to reveal their toxic mechanism to insects.


Assuntos
Substâncias Perigosas/toxicidade , Líquidos Iônicos/toxicidade , Animais , Ânions/química , Bombyx/metabolismo , Bombyx/fisiologia , Catalase/metabolismo , Cátions , Cloretos , Estudos de Viabilidade , Imidazóis , Larva/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído , Superóxido Dismutase/metabolismo , Testes de Toxicidade
12.
Arch Insect Biochem Physiol ; 105(3): e21741, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33002240

RESUMO

Apoptosis is a process of programmed cell death that is regulated by genes independently. The Bm30kc6 gene is a kind of small molecular lipoprotein about 30 kDa, expressed highly in the late stage of the silkworm hemolymph. Our study showed that overexpression of Bm30kc6 could decrease caspase-3 activation. Meanwhile, activation of caspase-3 increased when Bm30kc6 expression was disturbed by small interfering RNA (siRNA). Cell apoptosis was decreased when Bm30kc6 was overexpressed under UV treatment. The apoptosis rate induced by actinomycin D is similar to the trend by UV. It was inferred that Bm30kc6 has an inhibitory effect on the apoptosis of silkworm cells. The apoptosis-related genes, such as BmFadd, BmDredd, and BmDaxx were increased after overexpression of Bm30kc6 or decreased after interference of siRNA. It was speculated that there was an interactive relationship between Bm30kc6, BmDaxx, BmFadd, and BmDredd in the apoptosis signaling pathways. We investigated the transcription expression of the Bm30kc6 gene in different growth stages and tissues of the silkworm. The results showed that Bm30kc6 reached its peak in the hemolymph during the 6th to 7th days of the 5th instar, or in spinning post 24 h of the silk gland. In the silkworm BmN cells treated with caspase-3/7 inhibitor, the caspase-3 enzyme activity, and the expression levels of Bm30kc6, BmFadd, BmDredd, and BmDaxx were significantly reduced. The expression levels of Bm30kc6 increased sharply when silkworms were treated by molting hormone at Day 3 or 5 of the 5th instar. The results indicated that the expression of the Bm30kc6 gene was affected by the molting hormone and was likely to be its downstream target. In conclusion, the results suggest that the Bm30kc6 gene is involved in the regulation of the apoptotic signaling pathway and plays a role in the apoptotic process.


Assuntos
Apoptose/genética , Bombyx/crescimento & desenvolvimento , Bombyx/genética , Animais , Bombyx/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Inibidores de Caspase/farmacologia , Dactinomicina/farmacologia , Ecdisona/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Hemolinfa/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Raios Ultravioleta
13.
Arch Insect Biochem Physiol ; 105(3): e21737, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32926465

RESUMO

Olfaction plays an essential role in feeding and information exchange in insects. Previous studies on the olfaction of silkworms have provided a wealth of information about genes and proteins, yet, most studies have only focused on a single gene or protein related to the insect's olfaction. The aim of the current study is to determine key proteins in the olfactory system of the silkworm, and further understand protein-protein interactions (PPIs) in the olfactory system of Lepidoptera. To achieve this goal, we integrated Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses. Furthermore, we selected 585 olfactory-related proteins and constructed a (PPI) network for the olfactory system of the silkworm. Network analysis led to the identification of several key proteins, including GSTz1, LOC733095, BGIBMGA002169-TA, BGIBMGA010939-TA, GSTs2, GSTd2, Or-2, and BGIBMGA013255-TA. A comprehensive evaluation of the proteins showed that glutathione S-transferases (GSTs) had the highest ranking. GSTs also had the highest enrichment levels in GO and KEGG. In conclusion, our analysis showed that key nodes in the biological network had a significant impact on the network, and the key proteins identified via network analysis could serve as new research targets to determine their functions in olfaction.


Assuntos
Bombyx/genética , Proteínas de Insetos/genética , Mapas de Interação de Proteínas , Olfato , Animais , Bombyx/metabolismo , Glutationa Transferase/metabolismo , Proteínas de Insetos/metabolismo , Olfato/genética
14.
Chemosphere ; 259: 127481, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32650163

RESUMO

Zinc Oxide nanoparticles (ZnO NPs) has been heavily used in the industry, and increasing concerns on the ecotoxicity has arisen due to the risk of release into the environment. In this work, silkworm was used here as a model organism to study the toxicity of ZnO NPs, due to the presence of a conserved immune response as well as a pharmacokinetics similar to mammals. Zn accumulation, biodistribution and toxicity in silkworms were monitored at different time points after a subcutaneous injection. The highest cumulative content of ZnO NPs was detected in the midgut. The results of catalytic activity studies confirmed that the antioxidant enzymes (SOD, CAT, GSH-PX) in midgut cells were expressed in response to ZnO NPs. The expression of genes (Dronc and Caspase-1) related to apoptosis was increased, while the Trt gene was down-regulated. A possible mechanism was proposed for toxicity of ZnO NPs to silkworms.


Assuntos
Bombyx/fisiologia , Nanopartículas Metálicas/toxicidade , Óxido de Zinco/toxicidade , Animais , Antioxidantes/metabolismo , Apoptose , Bombyx/metabolismo , Nanopartículas , Distribuição Tecidual , Testes de Toxicidade Aguda
15.
Arch Insect Biochem Physiol ; 105(1): e21724, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32623793

RESUMO

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen in silkworm, and the molecular mechanism of B. mori defense to BmNPV infection is still unclear. RNA interference (RNAi) is well-known as an intracellular conserved mechanism that is critical in gene regulation and cell defense. The antiviral RNAi pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs that guide the recognition and cleavage of complementary viral target RNAs. In this study, a Dicer-2 (Dcr2) gene was identified in B. mori and its antiviral function was explored. Dcr2 messenger RNA (mRNA) expression was the highest in hemocytes and expressed in all stages of silkworm growth. After infection with BmNPV, the expression of Dcr2 mRNA was significantly increased after infection in midgut and hemocytes. The expression of Dcr2 was significantly upregulated by injecting dsRNA (dsBmSPH-1) into silkworm after 48 hr. Knocking down the expression level of Dcr2 using specific dsRNA in silkworm, which modestly enhanced the production of viral genomic DNA. Our results suggested that the Dcr2 gene in B. mori plays an important role in against BmNPV invasion.


Assuntos
Bombyx/genética , Interações Hospedeiro-Patógeno/genética , Proteínas de Insetos/genética , Nucleopoliedrovírus/fisiologia , Animais , Bombyx/metabolismo , Bombyx/virologia , Proteínas de Insetos/metabolismo , Filogenia , RNA Mensageiro/metabolismo , Análise de Sequência de DNA
16.
Gene ; 760: 144998, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32717304

RESUMO

The life cycle of holometabolous insects involves different stages and cathepsin plays an important role in insect metamorphosis. In the present study, we investigated the function of Bombyx mori cathepsin-L (Bm-CatL) during metamorphosis and analyzed their role in programmed cell death (PCD) of the fat body. The results showed that knockdown of Bm-CatL by RNA interference led to abnormal pupation and a delay in fat body degradation during metamorphosis. Furthermore, PCD inhibition was observed in the fat body after downregulation of Bm-CatL. To confirm this finding, PCD was induced in Bombyx mori embryonic (BmE) cells by ultraviolet ray irradiation. We found that the PCD of BmE cells was weakened after knocking down Bm-CatL. Moreover, overexpression of Bm-CatL in cells promoted PCD. Overall, our results showed that Bm-CatL is involved in the degradation of internal tissues and promotes the PCD of cells involved in the pupation of silkworms. Thus, this study provides us with a better understanding for function of cathepsin-L during metamorphosis.


Assuntos
Bombyx/fisiologia , Catepsina L/metabolismo , Tecido Adiposo/metabolismo , Animais , Apoptose/fisiologia , Bombyx/genética , Bombyx/metabolismo , Corpo Adiposo/metabolismo , Proteínas de Insetos/metabolismo , Metamorfose Biológica , Interferência de RNA
17.
PLoS Pathog ; 16(6): e1008633, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511266

RESUMO

DNA viruses can hijack and manipulate the host chromatin state to facilitate their infection. Multiple lines of evidences reveal that DNA virus infection results in the host chromatin relocation, yet there is little known about the effects of viral infection on the architecture of host chromatin. Here, a combination of epigenomic, transcriptomic and biochemical assays were conducted to investigate the temporal dynamics of chromatin accessibility in response to Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The high-quality ATAC-seq data indicated that progressive chromatin remodeling took place following BmNPV infection. Viral infection resulted in a more open chromatin architecture, along with the marginalization of host genome and nucleosome disassembly. Moreover, our results revealed that chromatin accessibility in uninfected cells was regulated by euchromatic modifications, whereas the viral-induced highly accessible chromatin regions were originally associated with facultative heterochromatic modification. Overall, our findings illustrate for the first time the organization and accessibility of host chromatin in BmNPV-infected cells, which lay the foundation for future studies on epigenomic regulation mediated by DNA viruses.


Assuntos
Baculoviridae/fisiologia , Bombyx , Eucromatina , Genoma de Inseto , Interações Hospedeiro-Patógeno , Animais , Bombyx/genética , Bombyx/metabolismo , Bombyx/virologia , Linhagem Celular , Eucromatina/genética , Eucromatina/metabolismo , Eucromatina/virologia
18.
PLoS Genet ; 16(6): e1008622, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520935

RESUMO

Insect courtship and mating depend on integration of olfactory, visual, and tactile cues. Compared to other insects, Bombyx mori, the domesticated silkworm, has relatively simple sexual behaviors as it cannot fly. Here by using CRISPR/Cas9 and electrophysiological techniques we found that courtship and mating behaviors are regulated in male silk moths by mutating genes in the sex determination cascade belonging to two conserved pathways. Loss of Bmdsx gene expression significantly reduced the peripheral perception of the major pheromone component bombykol by reducing expression of the product of the BmOR1 gene which completely blocked courtship in adult males. Interestingly, we found that mating behavior was regulated independently by another sexual differentiation gene, Bmfru. Loss of Bmfru completely blocked mating, but males displayed normal courtship behavior. Lack of Bmfru expression significantly reduced the perception of the minor pheromone component bombykal due to the down regulation of BmOR3 expression; further, functional analysis revealed that loss of the product of BmOR3 played a key role in terminating male mating behavior. Our results suggest that Bmdsx and Bmfru are at the base of the two primary pathways that regulate olfactory-based sexual behavior.


Assuntos
Bombyx/genética , Genes de Insetos , Preferência de Acasalamento Animal , Atrativos Sexuais/metabolismo , Processos de Determinação Sexual/genética , Animais , Bombyx/metabolismo , Bombyx/fisiologia , Feminino , Masculino , Receptores de Feromonas/genética , Receptores de Feromonas/metabolismo , Atrativos Sexuais/genética , Olfato
19.
J Insect Sci ; 20(3)2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32556319

RESUMO

Bombyx mori vitellogenin (BmVg) is highly upregulated during pupation, and the 20-hydroxyecdysone and amino acids may regulate stage-specific BmVg expression. However, previous studies showed that other factors may also affect stage-specific BmVg expression. Here, we characterized effective BmVg transcription factors by identifying the corresponding cis-regulatory elements (CREs). We prepared transgenic B. mori, in which DsRed was driven by various lengths of BmVg promoter. qRT-PCR analysis showed that DsRed expression driven by a 1.0-kb BmVg promoter (VgP1.0K) was consistent with endogenous BmVg. VgP1.0K specificity was closer to the endogenous BmVg promoter than that of VgP0.8K. These results suggest that CREs affecting stage-specific BmVg expression were localized to the 1.0-kb BmVg promoter. We investigated the effects of certain CREs that could influence the stage specificity of BmVg promoter on BmVg expression in transgenic B. mori. The relative DsRed expression was significantly reduced in transgenic female B. mori and the peak in DsRed expression was delayed after E-box CRE mutation. These results demonstrate that the E-box element enhanced BmVg expression and also affected stage-specific BmVg expression. Moreover, the relative DsRed expression was significantly increased in transgenic female of B. mori after 3×BD CRE mutation in BmVg promoter. However, the stage specificity of the mutated promoter was consistent with that of the endogenous BmVg promoter. The 3×BD element downregulated BmVg but had no effect on stage-specific BmVg expression. The present study promoted the process of elucidating the regulatory network for stage-specific BmVg expression and furnished a theoretical basis for the application of BmVg promoter.


Assuntos
Bombyx/genética , Expressão Gênica/genética , Genes de Insetos , Proteínas de Insetos/genética , Elementos Reguladores de Transcrição/genética , Fatores de Transcrição/genética , Vitelogeninas/genética , Animais , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Vitelogeninas/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(17): 9292-9301, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32277029

RESUMO

In insects, 20-hydroxyecdysone (20E) limits the growth period by triggering developmental transitions; 20E also modulates the growth rate by antagonizing insulin/insulin-like growth factor signaling (IIS). Previous work has shown that 20E cross-talks with IIS, but the underlying molecular mechanisms are not fully understood. Here we found that, in both the silkworm Bombyx mori and the fruit fly Drosophila melanogaster, 20E antagonized IIS through the AMP-activated protein kinase (AMPK)-protein phosphatase 2A (PP2A) axis in the fat body and suppressed the growth rate. During Bombyx larval molt or Drosophila pupariation, high levels of 20E activate AMPK, a molecular sensor that maintains energy homeostasis in the insect fat body. In turn, AMPK activates PP2A, which further dephosphorylates insulin receptor and protein kinase B (AKT), thus inhibiting IIS. Activation of the AMPK-PP2A axis and inhibition of IIS in the Drosophila fat body reduced food consumption, resulting in the restriction of growth rate and body weight. Overall, our study revealed an important mechanism by which 20E antagonizes IIS in the insect fat body to restrict the larval growth rate, thereby expanding our understanding of the comprehensive regulatory mechanisms of final body size in animals.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Tamanho Corporal/fisiologia , Proteína Fosfatase 2/metabolismo , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Ecdisterona/metabolismo , Corpo Adiposo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Insetos/genética , Insetos/crescimento & desenvolvimento , Insetos/metabolismo , Insulina/metabolismo , Larva/crescimento & desenvolvimento , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Somatomedinas/metabolismo
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