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1.
Front Cell Infect Microbiol ; 12: 1026154, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36304932

RESUMO

Nosema ceranae is a honey bee gut parasite that has recently spilled to another honey bee host through trading. The impact of infection on the native host is minor, which is substantial in the novel host. In this study, artificial inoculation simulated the parasite transmission from the native to the novel host. We found that the parasite initiated proliferation earlier in the novel host than in the native host. Additionally, parasite gene expression was significantly higher when infecting the novel host compared with the native host, leading to a significantly higher number of spores. Allele frequencies were similar for spores of parasites infecting both native and novel hosts. This suggests that the high number of spores found in the novel host was not caused by a subset of more fit spores from native hosts. Native hosts also showed a higher number of up-regulated genes in response to infection when compared with novel hosts. Our data further showed that native hosts suppressed parasite gene expression and arguably sacrificed cells to limit the parasite. The results provide novel insights into host defenses and gene selection during a parasite spillover event.


Assuntos
Nosema , Parasitos , Abelhas , Animais , Parasitos/genética , Nosema/genética , Genoma
2.
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
3.
Front Cell Infect Microbiol ; 12: 897509, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36046739

RESUMO

Microsporidia are obligate intracellular parasites and possess a unique way of invading hosts, namely germination. Microsporidia are able to infect almost all animal cells by germination. During the process, the polar tube extrudes from the spores within, thus injecting infectious sporoplasm into the host cells. Previous studies indicated that subtilisin-like protease 1 (NbSLP1) of microsporidia Nosema bombycis were located at the polar cap of germinated spores where the polar tube extrusion. We hypothesized that NbSLP1 is an essential player in the germination process. Normally, SLP need to be activated by autoproteolysis under conditions. In this study, we found that the signal peptide of NbSLP1 affected the activation of protease, two self-cleavage sites were involved in NbSLP1 maturation between Ala104Asp105 and Ala124Asp125 respectively. Mutants at catalytic triad of NbSLP1 confirmed the decreasing of autoproteolysis. This study demonstrates that intramolecular proteolysis is required for NbSLP1 maturation. The protease undergoes a series of sequential N-terminal cleavage events to generate the mature enzyme. Like other subtilisin-like enzymes, catalytic triad of NbSLP1 are significant for the self-activation of NbSLP1. In conclusion, clarifying the maturation of NbSLP1 will be valuable for understanding the polar tube ejection mechanism of germination.


Assuntos
Proteínas Fúngicas , Nosema , Animais , Proteínas Fúngicas/genética , Nosema/genética , Esporos Fúngicos , Subtilisina/genética
4.
Sci Rep ; 12(1): 14406, 2022 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-36002552

RESUMO

The intracellular microsporidian parasite Nosema ceranae is known to compromise bee health by induction of energetic stress and downregulation of the immune system. Porphyrins are candidate therapeutic agents for controlling Nosema infection without adverse effects on honeybees. In the present work, the impact of two protoporphyrin IX derivatives, i.e. PP[Asp]2 and PP[Lys]2, on Apis mellifera humoral immune response has been investigated in laboratory conditions in non-infected and N. ceranae-infected honeybees. Fluorescence spectroscopy analysis of hemolymph showed for the first time that porphyrin molecules penetrate into the hemocoel of honeybees. Phenoloxidase (PO) activity and the expression of genes encoding antimicrobial peptides (AMPs: abaecin, defensin, and hymenoptaecin) were assessed. Porphyrins significantly increased the phenoloxidase activity in healthy honeybees but did not increase the expression of AMP genes. Compared with the control bees, the hemolymph of non-infected bees treated with porphyrins had an 11.3- and 6.1-fold higher level of PO activity after the 24- and 48-h porphyrin administration, respectively. Notably, there was a significant inverse correlation between the PO activity and the AMP gene expression level (r = - 0.61696, p = 0.0143). The PO activity profile in the infected bees was completely opposite to that in the healthy bees (r = - 0.5118, p = 0.000), which was related to the changing load of N. ceranae spores in the porphyrin treated-bees. On day 12 post-infection, the spore loads in the infected porphyrin-fed individuals significantly decreased by 74%, compared with the control bees. Our findings show involvement of the honeybee immune system in the porphyrin-based control of Nosema infection. This allows the infected bees to improve their lifespan considerably by choosing an optimal PO activity/AMP expression variant to cope with the varying level of N. ceranae infection.


Assuntos
Nosema , Protoporfirinas , Monofosfato de Adenosina/farmacologia , Amidas/farmacologia , Animais , Abelhas , Imunidade , Monofenol Mono-Oxigenase , Nosema/fisiologia , Protoporfirinas/farmacologia
5.
J Invertebr Pathol ; 193: 107801, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35863438

RESUMO

Nosema ceranae is a microsporidium parasite that silently affects honey bees, causing a disease called nosemosis. This parasite produces resistant spores and germinates in the midgut of honey bees, extrudes a polar tubule that injects an infective sporoplasm in the host cell epithelium, proliferates, and produces intestinal disorders that shorten honey bee lifespan. The rapid extension of this disease has been reported to be widespread among adult bees, and treatments are less effective and counterproductive weakening colonies. This work aimed to evaluate the antifungal activity of a prototype formulation based on a non-toxic plant extract (HO21-F) against N. ceranae. In laboratory, honey bees were infected artificially, kept in cages for 17 days and samples were taken at 7 and 14 days post infection (dpi). At the same time, in field conditions we evaluated the therapeutic effect of HO21-F for 28 days in naturally infected colonies. The effectiveness of the treatment has been demonstrated by a reduction of 83.6 % of the infection levels observed in laboratory conditions at concentrations of 0.5 and 1 g/L without affecting the survival rate. Besides, in-field conditions we reported a reduction of 88 % of the infection level at a concentration of 2.5 g/L, obtaining better antifungal effectiveness in comparison to other commercially available treatments. As a result, we observed that the use of HO21-F led to an increase in population size and honey production, both parameters associated with colony strength. The reported antifungal activity of HO21-F against N. ceranae, with a significant control of spore proliferation in worker bees, suggests the promising commercial application use of this product against nosemosis, and it will encourage new research studies to understand the mechanism of action, whether related to the spore-inhibition effect and/or a stimulating effect in natural response of colonies to counteract the disease.


Assuntos
Microsporidiose , Nosema , Olea , Animais , Antifúngicos/farmacologia , Abelhas , Nosema/fisiologia , Extratos Vegetais/farmacologia
6.
Acta Parasitol ; 67(3): 1364-1371, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35857274

RESUMO

PURPOSE: Pebrine as the most dangerous disease of silkworm mostly caused by Nosema species has caused huge economic losses. There is no information on the species and the genomic sequences of the pebrine-causing microsporidia in Iran. METHODS: In the present research, we tried to determine the sequences of two regions of rDNA using molecular methods. First, infected larvae and mother moths were collected from several farms in the north of Iran for identification and molecular characterization of microsporidian isolates. After extracting the spores and genomic DNA from the collected samples, two fragments of internal transcribed spacer (ITS) rDNA and small subunit (SSU) rDNA were amplified and sequenced, and registered in NCBI database and then, the phylogenetic tree was drawn. RESULTS: Results showed the obtained sequences (ITS rDNA: Accession No. MZ322002 and SSU rDNA: Accession No. MZ314703) represent a new strain of Nosema bombycis, which differs from the sequences deposited in the NCBI. CONCLUSION: The new N. bombycis strain identified in our study will help in control and management of the pebrine disease by specific detection of the infectious agent.


Assuntos
Bombyx , Microsporidiose , Nosema , Animais , DNA Ribossômico/genética , Fazendas , Irã (Geográfico) , Microsporidiose/epidemiologia , Microsporidiose/veterinária , Nosema/genética , Filogenia , Esporos Fúngicos
7.
Sci Rep ; 12(1): 9326, 2022 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-35662256

RESUMO

Nosema ceranae is an intracellular parasite that infects honeybees' gut altering the digestive functions; therefore, it has the potential of affecting the composition of the gut microbiome. In this work, individual bees of known age were sampled both in spring and autumn, and their digestive tracts were assessed for N. ceranae infection. Intestinal microbiome was assessed by sequencing the bacterial 16S rRNA gene in two different gut sections, the anterior section (AS; midgut and a half of ileum) and the posterior section (PS; second half of ileum and rectum). A preliminary analysis with a first batch of samples (n = 42) showed that AS samples had a higher potential to discriminate between infected and non-infected bees than PS samples. As a consequence, AS samples were selected for subsequent analyses. When analyzing the whole set of AS samples (n = 158) no changes in α- or ß-diversity were observed between infected and non-infected bees. However, significant changes in the relative abundance of Proteobacteria and Firmicutes appeared when a subgroup of highly infected bees was compared to the group of non-infected bees. Seasonality and bees' age had a significant impact in shaping the bacteriome structure and composition of the bees' gut. Further research is needed to elucidate possible associations between the microbiome and N. ceranae infection in order to find efficient strategies for prevention of infections through modulation of bees' microbiome.


Assuntos
Microbioma Gastrointestinal , Nosema , Animais , Abelhas/genética , Nosema/genética , RNA Ribossômico 16S/genética , Estações do Ano
8.
J Exp Biol ; 225(13)2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35726829

RESUMO

Pollinators are exposed to numerous parasites and pathogens when foraging on flowers. These biological stressors may affect critical cognitive abilities required for foraging. Here, we tested whether exposure to Nosema ceranae, one of the most widespread parasites of honey bees also found in wild pollinators, impacts cognition in bumblebees. We investigated different forms of olfactory learning and memory using conditioning of the proboscis extension reflex. Seven days after being exposed to parasite spores, bumblebees showed lower performance in absolute, differential and reversal learning than controls. The consistent observations across different types of olfactory learning indicate a general negative effect of N. ceranae exposure that did not specifically target particular brain areas or neural processes. We discuss the potential mechanisms by which N. ceranae impairs bumblebee cognition and the broader consequences for populations of pollinators.


Assuntos
Nosema , Parasitos , Animais , Abelhas/parasitologia , Aprendizagem , Memória , Nosema/patogenicidade , Parasitos/patogenicidade , Olfato
9.
Exp Suppl ; 114: 137-152, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35544002

RESUMO

The microsporidium Nosema bombycis can infect and transmit both vertically and horizontally in multiple lepidopteran insects including silkworms and crop pests. While there have been several studies on the N. bombycis spore, there have been only limited studies on the N. bombycis sporoplasm. This chapter reviews what is known about this life cycle stage as well as published studies on purification of the N. bombycis sporoplasm and its survival in an in vitro cell culture system. Genetic transformation techniques have revolutionized the study of many pathogenic organisms. While progress has been made on the development of such systems for microsporidia, this critical problem has not been solved for these pathogens. This chapter provides a summary of the latest research progress on genetic manipulation of N. bombycis.


Assuntos
Bombyx , Nosema , Animais , Bombyx/genética , Técnicas Genéticas , Nosema/genética , Esporos Fúngicos/genética
10.
Exp Suppl ; 114: 153-177, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35544003

RESUMO

There has been increased focus on the role of microbial attack as a potential cause of recent declines in the health of the western honey bee, Apis mellifera. The Nosema species, N. apis and N. ceranae, are microsporidian parasites that are pathogenic to honey bees, and infection by these species has been implicated as a key factor in honey bee losses. Honey bees infected with both Nosema spp. display significant changes in their biology at the cellular, tissue, and organismal levels impacting host metabolism, immune function, physiology, and behavior. Infected individuals lead to colony dysfunction and can contribute to colony disease in some circumstances. The means through which parasite growth and tissue pathology in the midgut lead to the dramatic physiological and behavioral changes at the organismal level are only partially understood. In addition, we possess only a limited appreciation of the elements of the host environment that impact pathogen growth and development. Critical for answering these questions is a mechanistic understanding of the host and pathogen machinery responsible for host-pathogen interactions. A number of approaches are already being used to elucidate these mechanisms, and promising new tools may allow for gain- and loss-of-function experiments to accelerate future progress.


Assuntos
Nosema , Animais , Abelhas/genética , Interações Hospedeiro-Patógeno/genética , Insetos , Nosema/genética
11.
J Proteomics ; 263: 104617, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35595055

RESUMO

Microsporidium is a kind of intracellular fungal pathogen that greatly threatens the human health, breeding industry, and food security. All members of microsporidia possess a unique, highly specialized invasion organelle, described as the polar filament. Like "reversing a finger of gloves", the polar filament discharges out of mature spores to transform as the polar tube, and pathogenic sporoplasm is transported to host cell through polar tube to complete infection. During the invasion process, the structure of polar filament and polar tube has changed, so does the protein composition on them? In this study, we firstly proposed a purification method for polar filament and polar tube from microsporidium Nosema bombycis which was infected silkworm Bombyx mori, and it was also found that the structure of polar filament and polar tube was obviously different. Therefore, the proteome of these two structures was comparatively analyzed. A total of 881 and 1216 proteins were respectively identified from the polar filament and polar tube. Ten potential novel polar tube proteins (PTPs) were screened, providing a reference for the novel PTPs identification. Compared with the polar filament, there were 35 upregulated and 41 downregulated proteins on the polar tube. GO and KEGG pathway analysis of all proteins from the polar filament and polar tube provided us with a profound understanding for the microsporidian germination process, which was of great significance for clarifying the infection mechanism of microsporidia. SIGNIFICANCE: Microsporidia are obligate intracellular parasites that infect a wide variety of hosts, including humans. The polar filament is a unique invasion organelle for microsporidia, and it is also one of the important indexes of microsporidian taxonomy. The polar tube is deformed from the primitive polar filament in mature spores. During the germination, the polar filament turns into a polar tube, like "reversing a finger of gloves", through which pathogenic sporoplasm is transported to host cells to complete infection. Since the structure of the polar filament and polar tube has changed, what about their protein composition? In this study, it was the first time to purify the polar filament and the polar tube from microsporidium Nosema bombycis that was infected silkworm Bombyx mori, which provided new insights for studying the invasion organelle of microsporidia. Comparing the fine structure of polar filament and polar tube, we found that their structure was obviously different. Therefore, the protein composition of these two structures is supposed to be varied. In this case, the proteome of these two structures was comparatively analyzed. A total of 881 and 1216 proteins were respectively identified from the polar filament and polar tube. Ten potential novel polar tube proteins (PTPs) were screened, providing a reference for the novel PTPs identification. Compared with the polar filament, there were 35 upregulated and 41 downregulated proteins on the polar tube. GO and KEGG pathway analysis of all proteins from the polar filament and polar tube provided us with a profound understanding for the microsporidian germination process, which was of great significance for clarifying the infection mechanism of microsporidia.


Assuntos
Bombyx , Microsporídios não Classificados , Organelas , Proteoma , Animais , Bombyx/metabolismo , Bombyx/microbiologia , Proteínas Fúngicas/metabolismo , Microsporídios não Classificados/química , Microsporídios não Classificados/metabolismo , Nosema , Organelas/química , Organelas/metabolismo , Melhoramento Vegetal , Proteoma/metabolismo , Proteômica/métodos , Esporos Fúngicos/metabolismo
12.
Gene ; 834: 146607, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35609797

RESUMO

As a typical species of microsporidium, Nosema bombycis is the pathogen causing the pébrine disease of silkworm. Rapid proliferation of N. bombycis in host cells requires replication of genetic material. As eukaryotic origin recognition protein, origin recognition complex (ORC) plays an important role in regulating DNA replication, and Orc1 is a key subunit of the origin recognition complex. In this study, we identified the Orc1 in the microsporidian N. bombycis (NbOrc1) for the first time. The NbOrc1 gene contains a complete ORF of 987 bp in length that encodes a 328 amino acid polypeptide. Indirect immunofluorescence results showed that NbOrc1 were colocalized with Nbactin and NbSAS-6 in the nuclei of N. bombycis. Subsequently, we further identified the interaction between the NbOrc1 and Nbactin by CO-IP and Western blot. These results imply that Orc1 may be involved in the proliferation of the microsporidian N. bombycis through interacting with actin.


Assuntos
Bombyx , Nosema , Animais , Bombyx/genética , Bombyx/metabolismo , Nosema/genética , Nosema/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo
13.
Infect Genet Evol ; 102: 105309, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35636694

RESUMO

CCT is a chaperonin which is widely present in eukaryotic cells and mainly involves in the folding and assembly of cytoskeletal proteins ß-tubulin and actin. The alpha subunit of CCT(CCTα) plays a pivotal role in the folding and assembly of cytoskeletal protein(s) as an individuals or complexes. In this study, we report cloning, characterization and expression of the CCTα of Nosema bombycis (NbCCTα) for the first time. The NbCCTα gene contains a complete ORF of 1629 bp in length that encodes a 542-amino acid polypeptide. The NbCCTα is 59.662 kDa molecular weight in size with an isoelectric point (pI) of 5.81, no signal peptide or transmembrane domain. The IFA results showed that the NbCCTα was co-localized with actin and ß-tubulin in the cytoplasm, nucleus, nuclear membrane and plasma membrane of N. bombycis in the process of proliferation. qPCR analysis showed that the relative expression level of NbCCTα increased from 24 h to 96 h post-infection (hp.i) of N. bombycis, and reached the highest at 96 hp.i. The relative expression level of NbCCTα gene after RNAi was restrained at a low level from 48 hp.i to 96 hp.i. Knockdown of NbCCTα gene down-regulated the expression of Nbß-tubulin and Nbactin genes. These results imply that NbCCTα may play an important role in the lifecycle of N. bombycis.


Assuntos
Bombyx , Nosema , Actinas/metabolismo , Animais , Células Eucarióticas , Humanos , Nosema/genética , Tubulina (Proteína)/genética
14.
Parasit Vectors ; 15(1): 141, 2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35449112

RESUMO

BACKGROUND: Microsporidia are a group of intracellular parasitic eukaryotes, serious pathogens that cause widespread infection in humans, vertebrates, and invertebrates. Because microsporidia have a thick spore wall structure, the in vitro transformation, cell culture, and genetic operation technology of microsporidia are far behind that of other parasites. METHODS: In this study, according to an analysis of the life-cycle of microsporidia, Nosema bombycis, and different electro-transformation conditions, the transduction efficiency of introducing foreign genes into N. bombycis was systematically determined. RESULTS: We analyzed the direct electro-transformation of foreign genes into germinating N. bombycis using reporters under the regulation of different characteristic promoters. Furthermore, we systematically determined the efficiency of electro-transformation into N. bombycis under different electro-transformation conditions and different developmental stages through an analysis of the whole life-cycle of N. bombycis. These results revealed that foreign genes could be effectively introduced through a perforation voltage of 100 V pulsed for 15 ms during the period of N. bombycis sporeplasm proliferation. CONCLUSIONS: We present an effective method for electro-transformation of a plasmid encoding a fluorescent protein into N. bombycis, which provides new insight for establishing genetic modifications and potential applications in these intracellular parasites.


Assuntos
Bombyx , Nosema , Animais , Bombyx/metabolismo , Eletroporação , Humanos , Nosema/metabolismo , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo
15.
J Invertebr Pathol ; 191: 107755, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35405136

RESUMO

Secretion of hexokinase (HK) by microsporidia into infected cells suggests an important role for this enzyme for the intracellular development of parasites. To verify whether the expression of HK-specific antibodies in the host cell cytoplasm can suppress the growth of microsporidia, we constructed an immune library of recombinant scFv fragments against the enzyme of the honey bee pathogen Vairimorpha (Nosema) ceranae (VcHK) with a representativeness of about 5 million bacterial transformants. Two variants of VcHK-specific recombinant antibodies were selected by library panning and expressed in lepidopteran Sf9 cell line. Infecting of cells expressing two selected and control scFv fragments with V. ceranae spores was followed by their cultivation for 4 days. Analysis of parasite ß-tubulin as well as spore wall protein SWP32 transcripts in infected cultures by reverse transcription PCR and real-time qPCR showed (1) V. ceranae growth in cells heterologous to bee pathogens, (2) its inhibition by one of the selected VcHK-specific recombinant antibodies. The latter result once again emphasizes an important role of microsporidia hexokinases in their relationships with infected host cells and suggests further focusing on the mechanisms of such suppression, as well as on the search for new V. ceranae - inhibiting scFv fragments.


Assuntos
Nosema , Animais , Abelhas , Técnicas de Cultura de Células , Hexoquinase , Microsporídios , Nosema/fisiologia
16.
Bull Entomol Res ; 112(4): 502-508, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35382911

RESUMO

Nosema bombycis is a destructive and specific intracellular parasite of silkworm, which is extremely harmful to the silkworm industry. N. bombycis is considered as a quarantine pathogen of sericulture because of its long incubation period and horizontal and vertical transmission. Herein, two single-chain antibodies targeting N. bombycis hexokinase (NbHK) were cloned and expressed in fusion with the N-terminal of Slmb (a Drosophila melanogaster FBP), which contains the F-box domain. Western blotting demonstrated that Sf9-III cells expressed NSlmb-scFv-7A and NSlmb-scFv-6H, which recognized native NbHK. Subsequently, the NbHK was degraded by host ubiquitination system. When challenged with N. bombycis, the transfected Sf9-III cells exhibited better resistance relative to the controls, demonstrating that NbHK is a prospective target for parasite controls and this approach represents a potential solution for constructing N. bombycis-resistant Bombyx mori.


Assuntos
Bombyx , Nosema , Animais , Bombyx/genética , Drosophila melanogaster , Hexoquinase/metabolismo , Estudos Prospectivos
17.
Pest Manag Sci ; 78(6): 2215-2227, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35192238

RESUMO

BACKGROUND: Microsporidia, a group of obligate intracellular fungal-related parasites, have been used as efficient biocontrol agents for agriculture and forestry pests due to their host specificity and transovarial transmission. They mainly infect insect pests through the intestinal tract, but the interactions between microsporidia and the gut microbiota of the host have not been well demonstrated. RESULTS: Based on the microsporidia-Bombyx mori model, we report that the susceptibility of silkworms to exposure to the microsporidium Nosema bombycis was both dose and time dependent. Comparative analyses of the silkworm gut microbiome revealed substantially increased abundance of Enterococcus belonging to Firmicutes after N. bombycis infection. Furthermore, a bacterial strain (LX10) was obtained from the gut of B. mori and identified as Enterococcus faecalis based on 16S rRNA sequence analysis. E. faecalis LX10 reduced the N. bombycis spore germination rate and the infection efficiency in vitro and in vivo, as confirmed by bioassay tests and histopathological analyses. In addition, after simultaneous oral feeding with E. faecalis LX10 and N. bombycis, gene (Akirin, Cecropin A, Mesh, Ssk, DUOX and NOS) expression, hydrogen peroxide and nitric oxide levels, and glutathione S-transferase (GST) activity showed different degrees of recovery and correction compared with those under N. bombycis infection alone. Finally, the enterococcin LX protein was identified from sterile LX10 fermentation liquid based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. CONCLUSION: Altogether, the results revealed that E. faecalis LX10 with anti-N. bombycis activity might play an important role in protecting silkworms from microsporidia. Removal of these specific commensal bacteria with antibiotics and utilization of transgenic symbiotic systems may effectively improve the biocontrol value of microsporidia. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.


Assuntos
Bombyx , Nosema , Animais , Bombyx/metabolismo , Cromatografia Líquida , Enterococcus faecalis/genética , Nosema/genética , RNA Ribossômico 16S , Espectrometria de Massas em Tandem
18.
Proteomics ; 22(9): e2100224, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34997678

RESUMO

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful technology used to investigate the spatio-temporal distribution of a huge number of molecules throughout a body/tissue section. In this paper, we report the use of MALDI IMS to follow the molecular impact of an experimental infection of Apis mellifera with the microsporidia Nosema ceranae. We performed representative molecular mass fingerprints of selected tissues obtained by dissection. This was followed by MALDI IMS workflows optimization including specimen embedding and positioning as well as washing and matrix application. We recorded the local distribution of peptides/proteins within different tissues from experimentally infected versus non infected honeybees. As expected, a distinction in these molecular profiles between the two conditions was recorded from different anatomical sections of the gut tissue. More importantly, we observed differences in the molecular profiles in the brain, thoracic ganglia, hypopharyngeal glands, and hemolymph. We introduced MALDI IMS as an effective approach to monitor the impact of N. ceranae infection on A. mellifera. This opens perspectives for the discovery of molecular changes in peptides/proteins markers that could contribute to a better understanding of the impact of stressors and toxicity on different tissues of a bee in a single experiment.


Assuntos
Nosema , Animais , Abelhas , Biomarcadores , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
19.
Folia Microbiol (Praha) ; 67(3): 419-425, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35040074

RESUMO

In this study, NaYF4:20%Yb, 2%Er upconverting nanoparticles (UCNPs) were synthesized by solvothermal method and characterized by transmission electron microscopy and upconversion fluorescence spectrometry. The results showed that the UCNP particles present good dispersion and uniform spherical shape with a size of 29 ~ 42 nm. Hydroxyl UCNPs were converted to hydrophilic carboxylic acid-functionalized ones by ligand exchange, and the streptavidin was attached on the surface of carboxylic acid-functionalized UCNPs via amide bond. The DNA nanosensors based on UCNPs with DNA probes have been successfully developed. Only the genomic DNA of Nosema bombycis can be specifically detected by the DNA nanosensors when the DNA of Bombyx mori and its pathogens was used as target DNA. When the DNA nanosensors were used to detect the DNA of N. bombycis, a broad emission peak signal appeared at 580 nm. There is linear relationship between the signal intensity and DNA concentration of N. bombycis, I580/I545 (R2 = 0.820) and I545/I654 (R2 = 0.901). The detectable minimum concentration of genomic DNA of N. bombycis was 100 ng/µL while the tested concentrations of N. bombycis genomic DNA were 3000 ng/µL, 1500 ng/µL, 1000 ng/µL, 500 ng/µL, 250 ng/µL, and 100 ng/µL, respectively. The whole detection process for target DNA takes less than 60 min.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Nanopartículas , Ácidos Carboxílicos , DNA , Nanopartículas/química , Nosema
20.
Parasitol Res ; 121(1): 453-460, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34993633

RESUMO

Pebrine disease is caused by microporidia (Nosema bombycis) and is destructive to sericulture production. A carbendazim-based drug FangWeiLing (FWL) has a significant control effect on the disease, which is a successful example of drug treatment of microsporidia. In this study, the therapeutic effect and critical action time of FWL were investigated by silkworm rearing biological test. Besides, the hemolymph samples from silkworms in the control group, model group, and FWL group were analyzed by metabonomics based on gas chromatography-mass spectrometry (GC/MS). The results showed that FWL had a significant therapeutic effect on pebrine disease, and the critical action time was 24 ~ 48 h post inoculation. Forty-seven different metabolites related to pebrine disease were screened out, and correlated with starch and sucrose metabolism; aminoacyl-tRNA biosynthesis; arginine biosynthesis; glycine, serine, and threonine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis. After pretreatment with FWL, the metabolites were all effectively regulated, indicating productive intervention. Principal component analysis (PCA) also showed that the overall metabolic profile of the FWL group tended toward the control group. Compared with the control group, 16 different metabolites were obtained from the hemolymph of B.mori in FWL group, mainly involving aminoacyl-tRNA biosynthesis and taurine and hypotaurine metabolism. It indicated that FWL had some effect on silkworm metabolism, which might be related to the decrease in cocoon quality. In conclusion, combined with the life cycle of N. bombycis, the mechanism of carbendazim in the treatment of pebrine disease can be fully revealed. Carbendazim can effectively reduce the destruction of amino acid metabolism and carbohydrate metabolism by N. Bombycis infection by inhibiting the proliferation of the meronts in silkworms, thus maintaining the normal physiological state of B. mori and achieve therapeutic effects. GC/MS-based metabonomics is a valuable and promising strategy to understand the disease mechanism and drug treatment of pebrine disease.


Assuntos
Bombyx , Microsporidiose , Nosema , Animais , Benzimidazóis , Carbamatos , Cromatografia Gasosa-Espectrometria de Massas , Metabolômica
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