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Microsporidia are obligate intracellular eukaryotic parasites that have significantly reduced genomes and that have lost most of their introns. In the current study, we characterized a gene in microsporidia Nosema bombycis, annotated as TRAPα (HNbTRAPα). The homologous of TRAPα are a functional component of ER translocon and facilitates the initiation of protein translocation in a substrate-specific manner, which is conserved in animals but absent from most fungi. The coding sequence of HNbTRAPα consists of 2226 nucleotides, longer than the majority of homologs in microsporidia. A 3′ RACE analysis indicated that there were two mRNA isoforms resulting from non-canonical alternative polyadenylation (APA), and the polyadenylate tail was synthesized after the C951 or C1167 nucleotide, respectively. Indirect immunofluorescence analysis showed two different localization characteristics of HNbTRAPα, which are mainly located around the nuclear throughout the proliferation stage and co-localized with the nuclear in mature spores. This study demonstrated that the post-transcriptional regulation mechanism exists in Microsporidia and expands the mRNA isoform repertoire.

Microsporidia are ubiquitous in the environment, infecting almost all invertebrates, vertebrates, and some protists. The microsporidian Nosema bombycis causes silkworms pébrine disease and leads to huge economic losses. Parasite secreted proteins play vital roles in pathogen–host interactions. Serine protease inhibitors (serpins), belonging to the largest and most broadly distributed protease inhibitor superfamily, are also found in Microsporidia. In this study, we characterized 19 serpins (NbSPNs) in N. bombycis; eight of them were predicted with signal peptides. All NbSPN proteins contain a typical conserved serpin (PF00079) domain. The comparative genomic analysis revealed that microsporidia serpins were only found in the genus Nosema. In addition to N. bombycis, a total of 34 serpins were identified in another six species of Nosema including N. antheraeae (11), N. granulosis (8), Nosema sp. YNPr (3), Nosema sp. PM-1 (3), N. apis (4), and N. ceranae (5). Serpin gene duplications in tandem obviously occurred in Nosema antheranae. Notably, the NbSPNs were phylogenetically clustered with serpins from the Chordopoxvirinae, the subfamily of Poxvirus. All 19 NbSPN transcripts were detected in the infected midgut and fat body, while 19 NbSPN genes except for NbSPN12 were found in the transcriptome of the infected silkworm embryonic cell line BmE-SWU1. Our work paves the way for further study of serpin function in microsporidia.

Background: Microsporidia are a group of pathogens that infect all kinds of animals, such as humans, silkworms, honeybees, and shrimp; they, therefore, pose a severe threat to public health and the economy. There are over 1500 species of microsporidia that have been reported, among which Encephalitozoon hellem and Nosema bombycis are the representative zoonotic and insect-infecting species, respectively. Investigating their cell infection patterns is of great significance for understanding their infection mechanisms. Methods: Specific probes were designed for the ribosomal RNA sequences of microsporidia. Fluorescence in situ hybridization (FISH) was used to trace the proliferation cycle of the pathogens in different cells. Results: Here, two rRNA large subunit gene (LSUrRNA) probes specifically labeling N. bombycis were obtained. The life cycle of N. bombycis in silkworm cells and E. hellem in three kinds of host cells was graphically drawn. N. bombycis meronts were first observed at 30 hours post-infection (hpi), and they began merogony. Sporonts were observed at 42 hpi, and the first entire proliferation cycle was completed at 48 hpi. The proliferation cycle of E. hellem in RK13 and HEK293 epithelial cells was almost the same, completing the first life cycle after 24 hpi, but it was significantly delayed to 32 hpi in RAW264.7. Conclusions: Specific FISH probes were established for labeling microsporidia in multiple host cells. The proliferation characteristics of representative zoonotic and insect-infecting microsporidian species were clarified. This study provides an experimental pattern for future analyses of microsporidian infection mechanisms.

Microsporidia are a group of obligate intracellular pathogens infecting nearly all animal phyla. The microsporidian Nosema bombycis has been isolated from several lepidopteran species, including the economy-important silkworms as well as several crop pests. Proteins secreted by parasites can be important virulent factors in modulating host pathways. Ricin is a two-chain lectin best known for its extreme vertebrate toxicity. Ricin B lectin-like proteins are widely distributed in microsporidia, especially in N. bombycis. In this study, we identify 52 Ricin B lectin-like proteins (RBLs) in N. bombycis. We show that the N. bombycis RBLs (NbRBLs) are classified into four subfamilies. The subfamily 1 was the most conserved, with all members having a Ricin B lectin domain and most members containing a signal peptide. The other three subfamilies were less conserved, and even lost the Ricin B lectin domain, suggesting that NbRBLs might be a multi-functional family. Our study here indicated that the NbRBL family had evolved by producing tandem duplications firstly and then expanded by segmental duplications, resulting in concentrated localizations mainly in three genomic regions. Moreover, based on RNA-seq data, we found that several Nbrbls were highly expressed during infection. Further, the results show that the NbRBL28 was secreted into host nucleus, where it promotes the expressions of genes involved in cell cycle progression. In summary, the great copy number, high divergence, and concentrated genome distribution of the NbRBLs demonstrated that these proteins might be adaptively evolved and played a vital role in the multi-host N. bombycis.

Background Nosema bombycis is a unicellular eukaryotic pathogen of the silkworm, Bombyx mori, and is an economic and occupational hazard in the silkworm industry. Because of its long incubation period and horizontal and vertical transmission, it is subject to quarantine measures in sericulture production. The microsporidian life-cycle includes a dormant extracellular phase and intracellular proliferation phase, with the proliferation period being the most active period. This latter period lacks spore wall protection and may be the most susceptible stage for control. Methods In order to find suitable target for the selective breeding of N. bombycis-resistant silkworm strains, we screen highly expressed membrane proteins from the transcriptome data of N. bombycis. The subcellular localization of the candidate protein was verified by Indirect immunofluorescence analysis (IFA) and immunoelectron microscopy (IEM), and its role in N. bombycis proliferation was verified by RNAi. Results The N. bombycis protein (NBO_76g0014) was identified as a transmembrane protein and named NbTMP1. It is homologous with hypothetical proteins NGRA_1734 from Nosema granulosis. NbTMP1 has a transmembrane region of 23 amino acids at the N-terminus. Indirect immunofluorescence analysis (IFA) results suggest that NbTMP1 is secreted on the plasma membrane as the spores develop. Western blot and qRT-PCR analysis showed that NbTMP1 was expressed in all developmental stages of N. bombycis in infected cells and in the silkworm midgut. Downregulation of NbTMP1 expression resulted in significant inhibition of N. bombycis proliferation. Conclusions We confirmed that NbTMP1 is a membrane protein of N. bombycis. Reduction of the transcription level of NbTMP1 significantly inhibited N. bombycis proliferation, and this protein may be a target for the selective breeding of N. bombycis-resistant silkworm strains. Graphical Abstract

Background Microsporidians are opportunistic pathogens with a wide range of hosts, including invertebrates, vertebrates and even humans. Microsporidians possess a highly specialized invasion structure, the polar tube. When spores encounter an appropriate environmental stimulation, the polar tube rapidly everts out of the spore, forming a 50–500 µm hollow tube that serves as a conduit for sporoplasm passage into host cells. The polar tube is mainly composed of polar tube proteins (PTPs). So far, five major polar tube proteins have been isolated from microsporidians. Nosema bombycis, the first identified microsporidian, infects the economically important insect silkworm and causes heavy financial loss to the sericulture industry annually. Results A novel polar tube protein of N. bombycis (NbPTP6) was identified. NbPTP6 was rich in histidine (H) and serine (S), which contained a signal peptide of 16 amino acids at the N-terminus. NbPTP6 also had 6 potential O-glycosylation sites and 1 potential N-glycosylation site. The sequence alignment analysis revealed that NbPTP6 was homologous with uncharacterized proteins from other microsporidians (Encephalitozoon cuniculi, E. hellem and N. ceranae). Additionally, the NbPTP6 gene was expressed in mature N. bombycis spores. Indirect immunofluorescence analysis (IFA) result showed that NbPTP6 is localized on the whole polar tube of the germinated spores. Moreover, IFA, enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays results revealed that NbPTP6 had cell-binding ability. Conclusions Based on our results, we have confirmed that NbPTP6 is a novel microsporidian polar tube protein. This protein could adhere with the host cell surface, so we speculated it might play an important role in the process of microsporidian infection.

The fungus Nosema bombycis causes significant economic losses via parasitism of an economically important insect. MicroRNAs (miRNAs) play important roles in regulating host and parasite gene expression via mRNA degradation or by inhibiting protein translation. To investigate whether microRNA-like RNAs (milRNAs) regulate N. bombycis pathogenesis and to better understand the regulatory mechanisms underlying infection, we constructed small RNA libraries from N. bombycis hyphae during the schizont proliferation period. Eleven novel milRNAs were determined by RNA sequencing and stem-loop reverse transcriptase PCR (RT-PCR) assays. Moreover, a virulence-associated milRNA, Nb-milR8, was identified as critical for N. bombycis proliferation by binding and downregulating expression of its target gene, BmPEX16, in the host during infection. Silencing of Nb-milR8 or overexpression of the target BmPEX16 gene resulted in increased susceptibility of Bombyx mori to N. bombycis infection. Taken together, these results suggest that Nb-milR8 is an important virulence factor that acts as an effector to suppress host peroxidase metabolism, thereby facilitating N. bombycis proliferation. These results provide important novel insights into interactions between pathogenic fungi and their hosts. IMPORTANCE A thorough understanding of fungal pathogen adaptations is essential for treating fungal infections. Recent studies have suggested that the role of small RNAs expressed in fungal microsporidia genomes are important for elucidating the mechanisms of fungal infections. Here, we report 11 novel microRNA-like RNAs (milRNAs) from the fungal microsporidium Nosema bombycis and identified NB-milRNAs that adaptively regulate N. bombycis proliferation. In addition, we demonstrate that N. bombycis modulates small RNA (sRNA)-mediated infection by encoding an Nb-miR8 that downregulates the expression of the host peroxidase metabolism protein BmPEX16, which is essential for peroxisome membrane biogenesis and peroxisome assembly. These results significantly contribute to our understanding of the pathogenic mechanisms of fungi, and especially microsporidia, while providing important targets for genetical engineering-based treatment of microsporidia.

Microsporidia are ubiquitous fungi-related parasites infecting nearly all vertebrates and invertebrates. Microsporidian Nosema bombycis is a natural pathogen of multiple insects, including the silkworm and many agricultural and forest pests. N. bombycis can transovarially transmit in silkworm and cause huge economic losses to the sericulture. However, it remains unclear whether N. bombycis vertically transmits in the crop pests Spodoptera litura and Helicoverpa armigera. Here, we investigated the infection of N. bombycis in S. litura and H. armigera to illuminate its infectivity and transovarial transmission. In result, tissue examination with light microscopy revealed that the fat body, midgut, malpighian tubules, hemolymph, testis, and ovary were all infected in both pest pupae. Immunohistochemical analysis (IHA) of the ovariole showed that a large number of parasites in maturation and proliferation presented in follicle cell, nurse cell, and oocyte, suggesting that N. bombycis can infect and multiply in these cells and probably transovarially transmit to the next generations in both pests. Microscopic examination on the egg infection rate demonstrated that 50% and 38% of the S. litura and H. armigera eggs were congenitally infected, respectively. IHA of both eggs manifested numerous spores and proliferative pathogens in the oocyte, confirming that N. bombycis can invade into the female germ cell from the parent body. After hatching of the infected eggs, we detected the infection in offspring larvae and found large quantities of proliferative pathogens, confirming that N. bombycis can transovarially transmit in S. litura and H. armigera, and probably persists in both pest populations via congenital infection. In summary, our work, for the first time, proved that N. bombycis is able to vertically transmit in S. litura and H. armigera via infecting the oocyte in the parent, suggesting that N. bombycis could be a biological insecticide for controlling the population of crop pests.

Pebrine is a destructive disease that exhibits horizontal and vertical transmission and therefore it is the only mandatory quarantine item in sericulture. Here, a field-friendly loop-mediated isothermal amplification (FF-LAMP) method has been developed and validated for the rapid detection of Nosema bombycis, a causative agent of pebrine disease in silkworm, Bombyx mori. FF-LAMP primers were selected and designed for small ribosomal subunit gene and the assay was performed to detect the N. bombycis infection in silkworm. The FF-LAMP reaction was effective at 6 mM MgSO4, 1.4 mM dNTPs at 63 °C. The detection range of LAMP assay was found to be 101 dilutions of N. bombycis spores. Specificity of the primers was tested using DNA isolated from pebrine infected silkworm, pebrine free silkworm and pure N. bombycis by conventional PCR and FF-LAMP assay. Results revealed that the primers were specific to N. bombycis DNA. The FF-LAMP assay was validated in different basic silkworm seed farms with simultaneous microscopic examination of N. bombycis infection. This newly developed method is highly effective, specific, sensitive and rapid in detecting N. bombycis infection, eliminating the DNA purification steps and usage of sophisticated equipment. This method can be used in testing large number of samples making it field friendly method in sericulture industry., Invertebrate Survival Journal, Vol 18 No 1 (2021)

Microsporidia are a big group of single-celled obligate intracellular organisms infecting most animals and some protozoans. These minimalist eukaryotes lack numerous genes in metabolism and vesicle trafficking. Here, we demonstrated that the spore wall protein NbSWP12 of microsporidium Nosema bombycis belongs to Bin/Amphiphysin/Rvs (BAR) protein family and can specifically bind with phosphatidylinositol 3-phosphate [Ptdlns(3)P]. Since Ptdlns(3)P is involved in endosomal vesicle biogenesis and trafficking, we heterologous expressed NbSWP12 in yeast Saccharomyces cerevisiae and proved that NbSWP12 can target the cell membrane and endocytic vesicles. Nbswp12 transformed into Gvp36 (a BAR protein of S. cerevisiae) deletion mutant rescued the defect phenotype of vesicular traffic. This study identified a BAR protein function in vesicle genesis and sorting and provided clues for further understanding of how microsporidia internalize nutrients and metabolites during proliferation.

The microsporidium Nosema bombycis (Nb) causes pebrine, a fatal disease in sericulture. Nb is effectively killed by chlorine dioxide (ClO2); however, the precise killing mechanism remains unclear. We used laser tweezers Raman spectroscopy (LTRS) to monitor the action of ClO2on individual Nb spores in real time. Raman peaks of ClO2appeared in Nb spores, corresponding to decreased peaks of trehalose that gradually disappeared. A peak (1658 cm–1) corresponding to the protein α-helix significantly weakened while that (1668 cm–1) corresponding to irregular protein structures was enhanced; their intensities were negatively correlated in a certain time range and dependent on ClO2concentration. The intensities of peaks at 782 cm–1(nucleic acids) and 1004 cm–1(phenylalanine of protein) did not change evidently even under extremely high ClO2concentrations. Thus, ClO2rapidly permeates the Nb spore wall, changing the protein secondary structure to lose biological function and destroy permeability, causing trehalose to leak out. These effects are ClO2concentration-dependent, but no other obvious changes to biomacromolecules were detected. Single-cell analysis using LTRS is an effective method to monitor the action of chemical sporicides on microbes in real time, providing insight into the heterogeneity of cell stress resistance.

Once awoken from dormancy, the cellular matter of microsporidia is delivered directly into the host cell cytoplasm through the polar tube. This means that the microsporidia are difficult to study biologically in their active state without a contaminating signal from the host cell. Sporoplasm is a cell type of microsporidia in vitro, but relatively little attention has been paid to the sporoplasm in the past 150 years due to a lack of an effective separation method. Nosema bombycis, the first reported microsporidium, is a type of obligate intracellular parasite that infects silkworms and can be induced to germinate in alkaline solution in vitro. We successfully separated the N. bombycis sporoplasm in vitro, and the morphological and structural characteristics were investigated. These results provide important insight into the biology and pathogenesis of microsporidia and potentially provide a possible strategy for genetic manipulation of microsporidia targeting the sporoplasm., Microsporidia are obligate intracellular parasites that infect a wide variety of host organisms, including humans. The sporoplasm is the initial stage of microsporidian infection and proliferation, but its morphological and molecular characteristics are poorly understood. In this study, the sporoplasm of Nosema bombycis was successfully isolated and characterized after the induction of spore germination in vitro. The sporoplasm was spherical, 3.64 ± 0.41 μm in diameter, had the typical two nuclei, and was nonrefractive. Scanning and transmission electron microscopy analyses revealed that the sporoplasm was surrounded by a single membrane, and the cytoplasm was usually filled with relatively homogeneous granules, possibly ribosomes, and contained a vesicular structure comprising a concentric ring and coiled tubules. Propidium iodide staining revealed that the sporoplasm membrane showed stronger membrane permeability than did the cell plasma membrane. Transmission electron microscopy (TEM) revealed that the sporoplasm can gain entry to the host cell by phagocytosis. Transcriptome analysis of mature spores and sporoplasms showed that 541 significantly differentially expressed genes were screened (adjusted P value [Padj] < 0.05), of which 302 genes were upregulated and 239 genes were downregulated in the sporoplasm. The majority of the genes involved in trehalose synthesis metabolism, glycolysis, and the pentose phosphate pathway were downregulated, whereas 10 transporter genes were upregulated, suggesting that the sporoplasm may inhibit its own carbon metabolic activity and obtain the substances required for proliferation through transporter proteins. This study represents the first comprehensive and in-depth investigation of the sporoplasm at the morphological and molecular levels and provides novel insights into the biology of microsporidia and their infection mechanism. IMPORTANCE Once awoken from dormancy, the cellular matter of microsporidia is delivered directly into the host cell cytoplasm through the polar tube. This means that the microsporidia are difficult to study biologically in their active state without a contaminating signal from the host cell. Sporoplasm is a cell type of microsporidia in vitro, but relatively little attention has been paid to the sporoplasm in the past 150 years due to a lack of an effective separation method. Nosema bombycis, the first reported microsporidium, is a type of obligate intracellular parasite that infects silkworms and can be induced to germinate in alkaline solution in vitro. We successfully separated the N. bombycis sporoplasm in vitro, and the morphological and structural characteristics were investigated. These results provide important insight into the biology and pathogenesis of microsporidia and potentially provide a possible strategy for genetic manipulation of microsporidia targeting the sporoplasm.

Microsporidia Nosema bombycis CQ1 can be vertically transmitted in silkworm Bombyx mori but Vairimorpha necatrix BM cannot. Therefore, the pathological differences in silkworm infected with these two microsporidia required clarification. Here, we compared the virulence of N. bombycis CQ1 and V. necatrix BM against silkworm. The pathological characteristics in intestine, testis and ovary were surveyed using paraffin sections, scanning electron microscopy and transmission electron microscopy. Our data firstly showed that the virulence of V. necatrix BM was weaker than that of N. bombycis CQ1. Secondly, the typical symptom of V. necatrix BM infection is making xenomas, which are full of pathogens in different stages, at the posterior of intestine. However, no xenomas were formed surrounding intestines infected with N. bombycis CQ1. Thirdly, N. bombycis CQ1 can cluster spores near the trachea while infecting ovaries. It is worth noting that N. bombycis CQ1 infected epithelial cells and connective tissues of ovaries, while V. necatrix BM did not. Although silkworm ovaries can not be infected by V. necatrix BM in vivo, it can infect embryonic and ovarian cell lines in vitro. This study is the first report about comparing infection features of N. bombycis CQ1 and V. necatrix BM in silkworm tissues and it provided elaborate and visual information of pathological characteristics which can help to explain the different transmission strategies of these two microsporidia.

"Pe ' brine" is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the beta-tubulin gene in the pe ' brine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 x 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV)

Several species and strains of microsporidia have been isolated from infected silkworms among which pebrine caused by Nosema bombycis Nageli is the most important. Infection from this disease causes severe economic loss in sericulture. Reduction of larval and pupal development and reduced weights in silkworms due to infection has been reported. In the present study, five microsporidian ( Nosema) isolates from mulberry silkworm, Bombyx mori L. collected from different locations in West Bengal, India were sampled to study the impact of their infection on the growth and development of B. mori . The study revealed significant differences among the isolates in their ability to cause a reduction in the larval and pupal development of silkworm. Healthy larvae showed better body and tissue weights which were significantly higher than in infected lots. Among the isolates, M5 registered the maximum reduction in relative growth rate, larval silk gland tissue somatic index, larval male and female gonad tissue somatic index (GTSI) and pupal female GTSI compared to the healthy control. Male and female pupa treated with M5 spores died before emergence, suggesting that the M5 isolate was the most virulent.

Microsporidia are obligate intracellular parasites and cannot be cultured in vitro, which limits the use of current genetic engineering technologies on this pathogen. We isolated sporoplasms of Nosema bombycis to attempt to culture the pathogen in vitro. Cell-free medium was designed and successfully maintained the sporoplasms for 5 days. The sporoplasms were able to absorb ATP from the medium and DNA replicated during cultivation, although there was not a significant change in morphology and number of sporoplasms. Our study provides a strategy for in vitro cultivation and genetic manipulation of microsporidia. .

Microsporidiosis of the silkworm Bombyx mori is caused by the highly virulent parasite Nosema bombycis (Nageli). The infection can be deleterious due to horizontal and vertical transmission, causing heavy damage to the sericulture industry. In recent years, molecular diagnostics has revolutionized the possibility to detect diseases in terms of rapidity and simplicity, however, most of them are time consuming, require sophisticated instruments and skilled personnel. In this study, the polar tube protein 1 gene of N. bombycis (Indian isolate) was cloned, characterized and utilized for the development of rapid and simple loop mediated isothermal amplification assay (LAMP) for detection of microsporidiosis in silkworm B. mori. The LAMP reaction conditions were optimized to 65 °C for 60 min. The developed method demonstrated a higher sensitivity and the detection limit was found to be 2-fold higher than conventional PCR. This is the first report on loop mediated isothermal amplification assay that could be used to diagnose microsporidiosis at various developmental stages of the silkworm. This method serves as a robust alternative technique to conventional PCR and aids in the rapid diagnosis of N. bombycis infecting silkworm B. mori., Invertebrate Survival Journal, Vol 15 No 1 (2018)

Nosema bombycis contains functional aquaporins (NbAQPs), which are key targets for exploring the mechanism of N. bombycis infection; however, the regulation of these NbAQPs remains unknown. The two highly conserved asparagine-proline-alanine sequences (NPA motifs) play important roles in AQP biogenesis. As part of this study, we constructed a series of NbAQP mutants (NbAQP_NPA1, NbAQP_NPA2, and NbAQP_NPA1,2) and expressed them in BmN cells. The results showed that mutations in either NPA motif or in both NPA motifs did not affect NbAQP expression in vitro. After expression in Xenopus laevis oocytes, those injected with wild-type NbAQP rapidly expanded, whereas oocytes injected with NbAQP_NPAs did not significantly change in size. The associated water permeability (pf) of NbAQP_NPAs was significantly reduced five–six times compared to that of wild-type NbAQP. These results indicated that NPA motifs are necessary for the water channel function of AQPs in N. bombycis. The present study shows for the first time that the NbAQP NPA motif has an impact on the water permeability of aquaporin in N. bombycis, thereby providing a platform for further research into the mechanisms underlying the regulation of NbAQP expression.

Background Microsporidia are a group of eukaryotic intracellular parasites that infect almost all vertebrates and invertebrates. However, there is little information available of how microsporidia obtain nutrients and energy from host cells. The purpose of this study was to investigate the energy and material requirements of Nosema bombycis for the invasion procedure through analyzing the global variation of the gene expression, protein abundance, fatty acids level and ATP flux induced by the microsporidia N. bombycis infection in the midgut of the silkworm Bombyx mori. Methods A suppression subtractive hybridization (SSH) and quantitative real-time PCR (qPCR) analysis were performed to identify the genes upregulated in the midgut of B. mori 48 h following N. bombycis infection. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to annotate and summarize the differentially expressed genes, according to the categories ‘molecular function’, ‘cellular component’ and ‘biological process’. To evaluate the nutrition material and energy costs in B.mori infected by N. bombycis, biochemical analysis was performed to determine the variation of protein abundance, fatty acid levels and ATP flux with or without the microsporidia N. bombycis infection in the midgut of the silkworm B. mori. Results A total of 744 clones were obtained, 288 clones were randomly selected for sequencing, and 110 unigenes were generated. Amongst these, 49.21%, 30.16% and 14.29% genes were involved in 19 molecular functions, 19 biological processes and nine cellular components, respectively. A total of 11 oxidative phosphorylation- and eight proton-coupled ATP synthesis-related genes were upregulated. Seven protein degradation-, three fat degradation-related genes were upregulated, and no genes related to the de novo synthesis of amino acids and fatty acids were significantly upregulated. The data from the biochemical analysis showed the contents of total protein and ATP of B. mori midgut tissues decreased significantly, whereas the fatty acid content did not significantly change after four days of N. bombycis infection. Microsporidia N. bombycis infection upregulated the expression level of genes involved in host ATP synthesis, protein and fat degradation, which eventually causes the obvious decline of protein content and ATP synthesis in the host midgut, whereas the fatty acids content did not change significantly. Conclusions This study suggested to some extent that N. bombycis invasion can activate the host protein degradation and accelerate the production of host ATP. Microsporidia of N. bombycis show preference for proteins rather than fatty acids from the host to ensure the material preparation required by their parasitic life-cycle. Requirements of N. bombycis for energy were also mainly dependent on the host ATP production. This study provides a new data that may help our understanding of the molecular mechanisms of obtaining energy and nutrients from the host by the microsporidium N. bombycis. Electronic supplementary material The online version of this article (10.1186/s13071-018-2755-2) contains supplementary material, which is available to authorized users.

Pebrine disease is the only mandatory quarantine item in sericultural production due to its destructive consequences. So far, the mother moth microscopic examination method established by Pasteur (1870) remains the only detection method for screening for the causative agent Nosema bombycis (N. bombycis). Because pebrine is a horizontal and vertical transmission disease, it is better to inspect silkworm eggs and newly hatched larvae to investigate the infection rate, vertical transmission rate and spore load of the progenies. There is a rising demand for a more direct, effective and accurate detection approach in the sericultural industry. Here, we developed a molecular detection approach based on real-time quantitative PCR (qPCR) for pebrine inspection in single silkworm eggs and newly hatched larvae. Targeting the small-subunit rRNA gene of N. bombycis, this assay showed high sensitivity and reproducibility. Ten spores in a whole sample or 0.1 spore DNA (1 spore DNA represents the DNA content of one N. bombycis spore) in a reaction system was estimated as the detection limit of the isolation and real-time qPCR procedure. Silkworm egg tissues impact the detection sensitivity but are not significant in single silkworm egg detection. Of 400 samples produced by infected moths, 167 and 195 were scored positive by light microscopy and real-time qPCR analysis, respectively. With higher accuracy and the potential capability of high-throughput screening, this method is anticipated to be adaptable for pebrine inspection and surveillance in the sericultural industry. In addition, this method can be applied to ecology studies of N. bombycis-silkworm interactions due to its quantitative function.

The pathogen of pebrine is transmitted to a host either vertically or horizontally. Vertical transmission may arise in two distinct ways depending upon whether the passage of the pathogen occurs on the surface of the egg or within the ovary of the mother moth. Horizontal transmission occurs only in the larval stage. The most frequent source of infection in this stage is that which occurs when feces from infected larvae contaminate the food and are thus ingested by healthy larvae in the same colony. Sanitation and disease-free stock are the principal strategies of pebrine prevention.

Microsporidia is a group of obligatory intracellular parasites with a unique invasion mechanism. One of the highly specialized invasion structures is the polar tube which is an example of a biological nanotube the study of which could bring usable knowledge in biotechnology. The thesis is a literary overeview which deals with the structure of microsporidian spores and presents N. bombycis as a suitable representative for the study of the infectious apparatus. In more detail, it summarizes the background to the structure of the polar tube and wall of N. bombycis spore.

Infected beetles were collected and homogenized in sterile water. The homogenates were filtered through four layers of cheesecloth and centrifuged at 3000g for 15 min. The pellets were resuspended in sterile water, and the spores were purified by Percoll gradient centrifugation using 90% Percoll at 15,000g for 40 min. The spore band was collected and washed several times with sterile water. The purified spores (n = 50) were measured under a light microscope (IL/Leica Microsystems, Inc., Deerfield) with an ocular micrometer and photographed with the Microscope USB Camera.

Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during the loop mediated isothermal amplification (LAMP) procedure by using a pH meter. The genomic DNA of Nosema bombycis (N. bombycis) was amplified and detected by employing this LAMP-pH meter platform for the first time.

We successfully established a detection method which exhibited a markedly higher sensitivity than previously developed detection methods for Nosema bombycis by combining glass beads, FTA card, and LAMP. Spores of N. bombycis were first broken by acid-washed glass beads; the DNA was subsequently extracted and purified with the FTA card, and LAMP was performed using primers (LSU296) designed based on the sequence of the LSU rRNA of N. bombycis. The minimum detection concentration was 10 spores/mL. When this method was used to detect pebrine disease in silkworm egg, the detection rate for 500 silkworm eggs, in which only one egg was infected with N. bombycis, was 100 % under our optimized conditions. If the number of eggs in the sample increased to 800 or 1,000, the sample was divided into two equal portions, and the eggs were smashed with glass beads after the addition of 1 mL of TE buffer. The liquid in two tubes was later mixed and applied to the FTA card, and the detection rates were 100 %. Furthermore, the LAMP method established in our study could detect N. bombycis infection in silkworm 24 h earlier than microscopy.

The microsporidian Nosema bombycis is an obligate intracellular pathogen of the silkworm Bombyx mori, causing the epidemic disease Pebrine and extensive economic losses in sericulture. Although N. bombycis forms spores with rigid spore walls that protect against various environmental pressures, ingested spores germinate immediately under the extremely alkaline host gut condition (Lepidoptera gut pH > 10.5), which is a key developmental turning point from dormant state to infected state. However, to date this process remains poorly understood due to the complexity of the animal digestive tract and the lack of genetic tools for microsporidia. Here we show, using an in vitro spore germination model, how the proteome of N. bombycis changes during germination, analyse specific metabolic pathways employed in detail, and validate key functional proteins in vivo in silkworms. By a label-free quantitative proteomics approach that is directly based on high-resolution mass spectrometry (MS) data, a total of 1136 proteins were identified with high confidence, with 127 proteins being significantly changed in comparison to non-germinated spores. Among them, structural proteins including polar tube protein 1 and 3 and spore wall protein (SWP) 4 and 30 were found to be significantly down-regulated, but SWP9 significantly up-regulated. Some nucleases like polynucleotide kinase/phosphatase and flap endonucleases 1, together with a panel of hydrolases involved in protein degradation and RNA cleavage were overrepresented too upon germination, which implied that they might play important roles during spore germination. The differentially regulated trends of these genes were validated, respectively, by quantitative RT-PCR and 3 proteins of interest were confirmed by Western blotting analyses in vitro and in vivo. Furthermore, the pathway analysis showed that abundant up- and down-regulations appear involved in the glycolysis, pentose phosphate pathway, purine, and pyrimidine metabolism, suggesting preparations of energy generation and substance synthesis for the following invasion and proliferation inside the host. This report, to our knowledge, provides the first proteomic landscape of N. bombycis spores, and also a stepping stone on the way to further study of the unique infection mode of this economically important pathogen and other microsporidia in general.

The microsporidian Nosema bombycis is an obligate intracellular pathogen of the silkworm Bombyx mori, causing the epidemic disease Pebrine and extensive economic losses in sericulture. Although N. bombycis forms spores with rigid spore walls that protect against various environmental pressures, ingested spores germinate immediately under the extremely alkaline host gut condition (Lepidoptera gut pH > 10.5), which is a key developmental turning point from dormant state to infected state. However, to date this process remains poorly understood due to the complexity of the animal digestive tract and the lack of genetic tools for microsporidia. Here we show, using an in vitro spore germination model, how the proteome of N. bombycis changes during germination, analyse specific metabolic pathways employed in detail, and validate key functional proteins in vivo in silkworms. By a label-free quantitative proteomics approach that is directly based on high-resolution mass spectrometry (MS) data, a total of 1136 proteins were identified with high confidence, with 127 proteins being significantly changed in comparison to non-germinated spores. Among them, structural proteins including polar tube protein 1 and 3 and spore wall protein (SWP) 4 and 30 were found to be significantly down-regulated, but SWP9 significantly up-regulated. Some nucleases like polynucleotide kinase/phosphatase and flap endonucleases 1, together with a panel of hydrolases involved in protein degradation and RNA cleavage were overrepresented too upon germination, which implied that they might play important roles during spore germination. The differentially regulated trends of these genes were validated, respectively, by quantitative RT-PCR and 3 proteins of interest were confirmed by Western blotting analyses in vitro and in vivo. Furthermore, the pathway analysis showed that abundant up- and down-regulations appear involved in the glycolysis, pentose phosphate pathway, purine, and pyrimidine metabolism, suggesting preparations of energy generation and substance synthesis for the following invasion and proliferation inside the host. This report, to our knowledge, provides the first proteomic landscape of N. bombycis spores, and also a stepping stone on the way to further study of the unique infection mode of this economically important pathogen and other microsporidia in general.

Microsporidia are highly specialized obligate intracellular parasites that can infect a wide variety of animals ranging from protists to mammals. The classical concept of the parasite invasion into a host cell involves its polar tube acting as a needle-syringe system. However, recent studies show microsporidian spores can also gain access to host cells by phagocytosis. The present study investigated the phagocytic uptake process of causative agent of the pebrine disease, Nosema bombycis, in several insect cell lines. We observed KOH-treated spores and cold-storaged spores can be easily uptaken by all the studied cell types 4 h post inoculation. In contrast, large numbers of freshly recovered spores remained in the culture medium. To further investigate the intracellular fates of KOH-treated spores and cold-storaged spores, electron and fluorescence microscopy were performed. No intracellular germination or subsequent parasite development were observed. Intracellular spores can be detected in host cells by polyclonal antibody 7 d post inoculation, suggesting phagocytized N. bombycis could not be digested by these non-professional phagocytes. Our results suggest that, phagocytic uptake of N. bombycis spores might represent a defense mechanism of the host cells and the intact spore wall barrier enable freshly recovered spores to keep resistance to this mechanism.

Microsporidia are a group of intracelluar eukaryotic parasites, which can infected almost all animals, including human beings. Till now, no mitochodria but mitosome, a remnant of mitochondria was discovered in this phylum. We present here the mitochondrial pyruvate dehydrogenase El (PDH, including PDHα and PDHβ) of the microsporidian Nosema bombycis, the pathogen of silkworm pébrine. Compared with PDH of microsporidian Encephalitozoon cuniculi and Antonospora locustae, both subunits are conserved. The phylogeny indicated that both subunits are mitochondrial. The syntenic maps revealed the subunits organization of NbPDH is distributed in different scaffolds, similar to that of EcPDH but different with AIPDH, and the relationship between phylogeny tree and organization of PDH suggest that the AlPDH subunits organization is the ancestral style of microsporidia, and through the genome evolution, the reshuffling of the chromosome of microsporidia occurred, the adjacent style of ALPDHE1 organization changed, and the two subunits separated and located to different chromosomes in E. cuniculi. For N. bombycis and N. ceranae, they locate to different scaffolds. In order to determine NbPDH subcellular localizations, we prepared the polyclonal antibodies against NbPDH prokaryotic fusion proteins, and adopted the colloidal gold immunological electron microscopy, the expression signals of NbPDH were observed in spores however, the subcellular localization were not definited. In general, through comparison of three microsporidian PDH molecular phylogeny, subunits organization in chromosomes, localization indicated that PDH is an interesting marker in microsporidia evolution.

Contrairement a ce qu’on pouvait penser, la filature (des cocons) semble bien avoir precede la sericiculture (production des cocons), elle-meme precedee d’un siecle environ par le tissage (Fraisse, 1949). La filature etait pratiquee dans la region d’Anduze dans les dernieres annees du XIIIe siecle, date a partir de laquelle apparurent les premieres plantations de muriers et les premiers elevages. Henri IV (1553-1610), Richelieu (1585-1642) et Colbert (1619-1683)donnerent une vive impulsion a cette activite naissante de sericiculture dans les Cevennes. Mais la Provence et le Bas-Languedoc etaient alors en avance sur la region d’Ales (XIIIe et XIVe siecles). Cependant le terrible hiver de 1709 devait detruire une partie importante des châtaigneraies et compromettre les recoltes pour des annees. Cet accident meteorologique devait indirectement servir la sericiculture, car le fond des vallees se couvrit de muriers, a vegetation plus rapide que celle des châtaigniers. Malgre des crises passageres dues aux variations des cours des cocons, la sericiculture se developpa et la grande prosperite de celle-ci s’installa de 1820 a 1853 dans les Cevennes et la region d’Ales. La foire du 24 aout faisait d’Ales la capitale des Cevennes, mais aussi celle de la soie. Dans le seul arrondissement d’Ales en 1841, la production de cocons atteint plus de 6 millions de kilos et les filatures de la region produisent plus de 500 000 kg de soie grege. C’est en 1853 que la production nationale allait atteindre son apogee avec 26 millions de kilos. C’est alors que commence l’epidemie de pebrine (Encadre 1) dont nous allons parler plus precisement dans la suite, qui fit tomber la production de cocons a 4 millions de kilos en 1865.

Nosema bombycis is a pathogen causing pebrine disease of the mulberry silkworm, Bombyx mori. The disease spreads mainly through transovarian transmission of environmental spore and secondarily through contaminated food, rearing appliances, etc. by primary spores. Ultra-structure studies using Scanning and Transmission Electron Microscopy of the two spores revealed differences in the primary spore which contained a Short Polar Tube (ST) with a thin wall (< 200 nm), and the environmental spore which contained a Long Polar Tube (LT) with a thick wall (> 200 nm). It is observed that the yield of spore with LT is highest in female moths, whereas, it is spores with ST are highest in male moths. Besides ultra-structures, the development pattern of the two types of spores is also different. It is an interesting finding in the present study that, spores of N. bombycis produced two types of spores and multiplied in different gender under the influence of the host’s reproductive role and physiology for transmission of disease. The detailed study on ultra-structure of disporous N. bombycis in both the sexes of B. mori along with their development in the life cycle stages of silkworms with special reference to the inoculum concentration of spore is discussed.

The spore replication rate of the parasitic microsporidian, Nosema bombycis, was investigated in the pupal and adult stages of two popular tropical silkworm races of India, Pure Mysore (multivoltine race) and NB18 (bivoltine race). The rate was assessed in terms of spore yield per unit weight of tissue. In pupae, spore replication in the bivoltine race was higher than that in the multivoltine race. Spore replication in both races followed a logistic pattern. Spore production increased substantially with age of moths in both races. The cephalothoracic region of each race had the highest spore concentration, especially around the wing and wing muscles.

International audience; Nosema bombycis is the causative agent of the silkworm Bombyx mori pebrine disease which inflicts severe worldwide economical losses in sericulture. Little is known about host-parasite interactions at the molecular level for this spore-forming obligate intracellular parasite which belongs to the fungi-related Microsporidia phylum. Major microsporidian structural proteins from the spore wall (SW) and the polar tube (PT) are known to be involved in host invasion. We developed a proteomic-based approach to identify few N. bombycis proteins belonging to these cell structures. Protein extraction protocols were optimized and four N. bombycis spore protein extracts were compared by SDS-PAGE and 2-DE to establish complementary proteomic profiles. Three proteins were shown to be located at the parasite SW. Moreover, 17 polyclonal antibodies were raised against major N. bombycis proteins from all extracts, and three spots were shown to correspond to polar tube proteins (PTPs) by immunofluorescent assay and transmission electron microscopy immunocytochemistry on cryosections. Specific patterns for each PTP were obtained by MALDI-TOF-MS and MS/MS. Peptide sequence tags were deduced by de novo sequencing using Peaks Online and DeNovoX, then evaluated by MASCOT and SEQUEST searches. Identification parameters were higher than false-positive hits, strengthening our strategy that could be enlarged to a nongenomic context.

Nosema bombycis is a spore-forming obligate intracellular parasite which belongs to the fungi-related Microsporidia phylum. It is the causative agent of the silkworm Bombyx mori pebrine disease which inflicts severe worldwide economical losses in sericulture. Little is known about N. bombycis in vivo dissemination mechanism and its interactions with its natural host B. mori at the cellular or molecular level, despite the ongoing genome sequencing projects of both organisms. In this study, we investigate N. bombycis (Zhenjiang isolate ISC-ZJ) dissemination in per os infected B. mori larvae (Nistari). At different times post-infestation, larvae were harvested and B. mori larva tissues, hemocytes and hemolymph were analysed by histochemistry and immunocytochemistry microscopy (optic and transmission electron microscopy). This allowed us to document not only N. bombycis ISC-ZJ in vivo life cycle at the ultrastructural level, but also the hitherto unreported N. bombycis dissemination route in silkworm larvae. N. bombycis infestation first settles in the anterior epithelial midgut. The infection spreads to the muscles and tracheae closely surrounding the anterior midgut. Distant tissues and organs are then infected concomitantly with the apparition of the parasite in the hemolymph or internalized in hemocytes, before the systemic infestation with all tissues parasitized. Our results strongly suggest that hemolymph and hemocytes are vectors for the parasite dissemination in its host. Moreover the silkworm innate immune response, although existing -we identified N. bombycis melanization and its phagocytosis by B. mori hemocytes - seem to be strongly overtaken by the microsporidian development with underlying mechanisms still unknown. To further study the N. bombycis/B. mori interactions at the molecular level, we focused on major microsporidian structural proteins from the spore wall and the polar tube which are known to be involved in host invasion. We developed a proteomic-based approach to identify few N. bombycis proteins belonging to these cell structures. Protein extraction protocols were optimized and four N. bombycis spore protein extracts were compared by mono- and bi-dimensionnal electrophoresis to establish complementary proteomic profiles. Three proteins (71, 48 and 30 kDa) were shown to be located at the parasite spore wall using a polyclonal antibody raised against formaldehyde-fixed spores. Moreover seventeen polyclonal antibodies were raised against major N. bombycis proteins from all extracts, and three spots, spot PEL1 (54 kDa, pI 5.4), spot PEL2 (36 kDa, pI 10) and spot PEL3 (150 kDa, pI 7.1), were shown to correspond to polar tube proteins (PTPs) by IFA and transmission electron microscopy immunocytochemistry on cryosections. These PTPs were respectively named NbPTP1, NbPTP2 and NbPTP3 according to their global physio-chemical properties. Specific patterns for each PTP were obtained by MALDI-TOF-MS and MS/MS mass spectrometry. Peptide sequence tags were deduced by de novo sequencing using Peaks Online and DeNovoX, then evaluated by Mascot and Sequest searches. Identification parameters were higher than false-positive hits, strengthening our strategy that could be enlarged to a non-genomic context., Nosema bombycis est un parasite obligatoire intracellulaire et eukaryoitique microsporidia apparenté aux champignons. Cette microsporidie est l'agent responsable de la pébrine, maladie du ver à soie Bombyx mori qui inflige de sévères pertes économiques à la sériciculture mondiale. Nous avons étudié l'interactions N. bombycis-B. mori in vivo : l'infestation par N. bombycis démarre au niveau de l'épithélium intestinal antérieur, puis s'étend aux muscles et trachées adjacents. Les tissus plus distants sont ensuite infectés. Cependant, les réponses immune mélanisation et phagocytose, l'hémolymphe et les hémocytes sont les vecteurs de la dissémination de N. bombycis dans son hôte. Nous avons développé une approche protéomique pour identifier des protéines de tube polaire (PTP). Trois PTPs ont été caracterisés par immunocytochimie MET et MS/MS. Des motifs de séquence peptidique ont pu en être déduits par les programmes Peaks Online et DeNovoX, puis évalués par algorithmes Mascot et Sequest

—Silkworm larvae infected transovarially with Nosema bombycis were fed with an aqueous suspension of Bavistin (50% carbendazim w/w) at 2 and 3% concentrations. The treatments increased the survival of worms and reduced the pebrine infection in the lot significantly, although complete elimination of infection was not observed. Significant beneficial effects of the treatments at 2 and 3% were also observed with respect to larval, cocoon and cocoon shell weights and cocoon to shell ratio. However, treatments with Bavistin at 4% concentration produced an adverse effect on cocoon characters.

Actuellement, la pébrine, microsporidiose due à N. bombycis et affectant entre autres le Bombyx du Mûrier, continue à sévir dans bon nombre de pays. Or, au moment où la sériciculture se développe dans certains pays tropicaux, il importe de maîtriser parfaitement les conditions sanitaires d'élevage, aspect fondamental à respecter pour lutter efficacement contre cette maladie, qui se transmet en outre par voie transovarienne. D'autre part, les chercheurs portent actuellement particulièrement attention sur de nouveaux procédés de diagnostic plus rapides et plus fiables, sur la chimiothérapie et sur la création de lignées résistantes à la maladie.