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ATP-binding cassette (ABC) transporter proteins, one of the largest families of membrane transport proteins, participate in almost all biological processes and widely exist in living organisms. Microsporidia are intracellular parasites; they can reduce crop yields and pose a threat to human health. The ABC proteins are also present in microsporidia and play a critical role in their proliferation and energy transport. In this study, a novel ABC transporter protein of Nosema bombycis named NoboABCG1.3 was identified. The NoboABCG1.3 protein is comprised of 640 amino acids, which contain six transmembrane domains and one nucleotide-binding domain. After N. bombycis infection of cells or tissues, quantitative reverse transcription polymerase chain reaction analysis revealed a progressive elevation in the transcript levels of NoboABCG1.3. Downregulation of NoboABCG1.3 expression significantly inhibited N. bombycis proliferation. Subsequently, a transgenic cell line stably expressing an interfering fragment of NoboABCG1.3 was established, which exhibited extreme inhibition on the proliferation of N. bombycis. These findings indicate that NoboABCG1.3 plays a role in the proliferation of N. bombycis and holds promise as a target for developing N. bombycis-resistant silkworms. [ABSTRACT FROM AUTHOR]

Microsporidia are opportunistic fungal‐like pathogens that cause microsporidiosis, which results in significant economic losses and threatens public health. Infection of domesticated silkworms by the microsporidium Nosema bombycis causes pébrine disease, for which this species of microsporidia has received much attention. Research has been conducted extensively on this microsporidium over the past few decades to better understand its infection, transmission, host–parasite interaction, and detection. Several tools exist to study this species including the complete genome sequence of N. bombycis. In addition to the understanding of N. bombycis being important for the silkworm industry, this species has become a model organism for studying microsporidia. Research on biology of N. bombycis will contribute to the development of knowledge regarding microsporidia and potential antimicrosporidia drugs. Furthermore, this will provide insight into the molecular evolution and functioning of other fungal pathogens. [ABSTRACT FROM AUTHOR]

Pebrine disease, caused by Nosema bombycis (Nb) infection in silkworms, is a severe and long-standing disease that threatens sericulture. As parasitic pathogens, a complex relationship exists between microsporidia and their hosts at the mitochondrial level. Previous studies have found that the translocator protein (TSPO) is involved in various biological functions, such as membrane potential regulation, mitochondrial autophagy, immune responses, calcium ion channel regulation, and cell apoptosis. In the present study, we found that TSPO expression in silkworms (BmTSPO) was upregulated following Nb infection, leading to an increase in cytoplasmic calcium, adenosine triphosphate, and reactive oxygen species levels. Knockdown and overexpression of BmTSPO resulted in the promotion and inhibition of Nb proliferation, respectively. We also demonstrated that the overexpression of BmTSPO promotes host cell apoptosis and significantly increases the expression of genes involved in the immune deficiency and Janus kinase-signal transducer and the activator of the transcription pathways. These findings suggest that BmTSPO activates the innate immune signalling pathway in silkworms to regulate Nb proliferation. Targeting TSPO represents a promising approach for the development of new treatments for microsporidian infections. [ABSTRACT FROM AUTHOR]

Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription–quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6 h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell’s energy resources. [ABSTRACT FROM AUTHOR]

Background: The interaction networks between coding and non-coding RNAs (ncRNAs) including long non-coding RNA (lncRNA), covalently closed circular RNA (circRNA) and miRNA are significant to elucidate molecular processes of biological activities and interactions between host and pathogen. Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-feeding industry. However, little is known about ncRNAs that take place in the microsporidia congenital infection. Here we conducted whole-transcriptome RNA-Seq analyses to identify ncRNAs and regulatory networks for both N. bombycis and host including silkworm embryos and larvae during the microsporidia congenital infection. Results: A total of 4,171 mRNAs, 403 lncRNA, 62 circRNAs, and 284 miRNAs encoded by N. bombycis were identified, among which some differentially expressed genes formed cross-talk and are involved in N. bombycis proliferation and infection. For instance, a lncRNA/circRNA competing endogenous RNA (ceRNA) network including 18 lncRNAs, one circRNA, and 20 miRNAs was constructed to describe 14 key parasites genes regulation, such as polar tube protein 3 (PTP3), ricin-B-lectin, spore wall protein 4 (SWP4), and heat shock protein 90 (HSP90). Regarding host silkworm upon N. bombycis congenital infection, a total of 14,889 mRNAs, 3,038 lncRNAs, 19,039 circRNAs, and 3,413 miRNAs were predicted based on silkworm genome with many differentially expressed coding and non-coding genes during distinct developmental stages. Different species of RNAs form interacting network to modulate silkworm biological processes, such as growth, metamorphosis and immune responses. Furthermore, a lncRNA/circRNA ceRNA network consisting of 140 lncRNAs, five circRNA, and seven miRNAs are constructed hypothetically to describe eight key host genes regulation, such as Toll-6, Serpin-6, inducible nitric oxide synthase (iNOS) and Caspase-8. Notably, cross-species analyses indicate that parasite and host miRNAs play a vital role in pathogen-host interaction in the microsporidia congenital infection. Conclusion: This is the first comprehensive pan-transcriptome study inclusive of both N. bombycis and its host silkworm with a specific focus on the microsporidia congenital infection, and show that ncRNA-mediated regulation plays a vital role in the microsporidia congenital infection, which provides a new insight into understanding the basic biology of microsporidia and pathogen-host interaction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pebrine disease, caused by Nosema bombycis (N. bombycis), is the most important pathogen known to the silk industry. Historical evidence from several countries shows that the outbreaks of pebrine disease have largely caused the decline of the sericulture industry. Prevention is the first line to combat pebrine as a deadly disease in silkworm; however, no effective treatment has yet been presented to treat the disease. Many different methods have been used for detection of pebrine disease agent. This review focuses on the explanation and comparison of these methods, and describes their advantages and/or disadvantages. Also, it highlights the ongoing advances in diagnostic methods for N. bombycis that could enable efforts to halt this microsporidia infection. The detection methods are categorized as microscopic, immunological and nucleic acid-based approaches, each with priorities over the other methods; however, the suitability of each method depends on the available equipment in the laboratory, the mass of infection, and the speed and sensitivity of detection. The accessibility and economic efficiency are compared as well as the speed and the sensitivity for each method. Although, the light microscopy is the most common method for detection of N. bombycis , qPCR is the most preferred method for large data based on speed and sensitivity as well as early detection ability. [Display omitted] • To detect Nosema bombycis , light microscopy is the most widely available but least sensitive method. • Electron microscopy can be used for pebrine disease agent, though it is cumbersome. • While agglutination and ELISA methods are useful, RT-qPCR is currently the preferred detection method given its speed and sensitivity. [ABSTRACT FROM AUTHOR]

The sericulture industry faces a significant threat from the Pebrine disease of silkworms, caused by Nosema bombycis. Nonetheless, the current microscopic diagnostic methods can be time-consuming, labor-intensive, and lacking sensitivity and accuracy. Therefore, it is crucial to develop a novel detection approach that is efficient, highly sensitive, and low-cost. In this regard, the CRISPR/Cas system has the potential to be a fast, accurate, and highly specific method of detection. Herein, using a microplate reader, a portable fluorescence detection device, and test strips as signal output tools respectively, we have efficiently developed three rapid and facile visual detection methods for N. bombycis using a CRISPR/Cas13a system with conjugation of Recombinase polymerase amplification (RPA). We evaluated the sensitivity of this combined technology by comparing it with the positive plasmid standard and the genome standard of N. bombycis. Remarkably, the sensitivity of the CRISPR/Cas13a system for N. bombycis positive plasmid standard based on the microplate reader, portable fluorescence detection device, and test strips was 1 copy/μL, 10 copies/μL, and 1 copy/μL, respectively, while for the N. bombycis genome standards, the detection sensitivity was 10 fg/μL, 10 fg/μL, and 1 fg/μL, respectively. In addition, extensive evaluations have demonstrated that the established technology can accurately detect N. bombycis without cross-reactivity with other pathogens, ensuring a specificity rate of 100%. In brief, this study will provide a practical, efficient, and affordable method for early and rapid detection of N. bombycis in various settings. [Display omitted] • A novel, robust, and visual approach has been developed for detecting Nosema bombycis based on CRISPR/Cas13a system. • High sensitivity with 1 copy/μL and 1 fg/μL and good specificity. • Three signal output tools: a microplate reader, a portable fluorescence detection device, and test strips. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • N. bombycis infection affects the physiology, development and immunity of B. mori. • Nosema altered expression of host genes, of which 1888 genes were significant. • Cecropins, DUOX and PIRK play a vital role in regulating the IMD pathway. • Nosema infection downregulates PPAE gene and suppress the synthesis of eumelanin. The infection caused by Nosema bombycis often known as pebrine, is a devastating sericulture disease. The infection can be transmitted to the next generation through eggs laid by infected female Bombyx mori moths (transovarial) as well as with N. bombycis contaminated food (horizontal). Most diagnoses were carried out in the advanced stages of infection until the time that infection might spread to other healthy insects. Hence, early diagnosis of pebrine is of utmost importance to quarantine infected larvae from uninfected silkworm batches and stop further spread of the infection. The findings of our study provide an insight into how the silkworm larval host defence system was activated against early N. bombycis transovarial infection. The results obtained from transcriptome analysis of infected 2nd instar larvae revealed significant (adjusted P-value < 0.05) expression of 1888 genes of which 801 genes were found to be upregulated and 1087 genes were downregulated when compared with the control. Pathway analysis indicated activation of the immune deficiency (IMD) pathway, which shows a potential immune defence response against pebrine infection as well as suppression of the melanin synthesis pathway due to lower expression of prophenoloxidase activating enzyme (PPAE). Liquid chromatography mass spectrometry (LC-MS/MS) analysis of haemolymph from infected larvae shows the secretion of serpin binding protein of N. bombycis which might be involved in the suppression of the melanization pathway. Moreover, among the differentially expressed genes, we found that LPMC-61 , yellow-y , gasp and osiris 9 can be utilised as potential markers for early diagnosis of transovarial pebrine infection in B. mori. Physiological as well as biochemical roles and functions of many of the essential genes are yet to be established, and enlightened research will be required to characterize the products of these genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify the production capacity of six commercial silkworm Bombyx mori L. (Lepidoptera: Bombycidae) lines (numbers 31, 32, 103, 104, 153, and 154), which were contaminated with Nosema bombycis Nageli spores (103 or 105 per ml), the fungus responsible for pebrine (pepper disease) in silkworms. Effects of the two fungal Microsporidia concentrations were tested on silkworm feeding on either treated (with 1.0% Lactobacillus rhamnosus probiotics or 0.5% Echinacea purpurea plant extract) or non‐treated (control) mulberry leaves (Moraceae). Traits included larval weight, cocoon weight, cocoon shell weight, pupal rate, silk shell percentage, total cocoon number, cocoon yield per 10 000 larvae, larval mortality, and incidence of infection in moth. Results showed that lines, feed supplements, and level of Microsporidia spore infection had significant effects on most estimated parameters. Both concentrations of Microsporidia spores led to a decrease in the silkworm performance and an increase in mortality. Supplementation of mulberry leaves with probiotics or plant extract resulted in improvement of economic traits in B. mori. Commercial line 153 performed the best in most of the studied traits and was highlighted as the optimum line for breeding in Iranian silkworm industry. Also, line 104 was the most resistant line to N. bombycis. This information will be helpful for selection of the best line for breeders according to the rearing conditions and desirable economic traits. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

As one of the core framework proteins of nuclear pore complex (NPC), nucleoporin Nupl70 acts as a structural adapter between the nucleolus and nuclear pore membrane and maintains the stability of NPC structure through interaction with other proteins. In this study, we identified a Nup170 protein in the microsporidian Nosema bombycis for the first time and named it as NbNup170. Secondary structure prediction showed that the NbNup170 contains α-helices and random coils. The three-dimensional structure of NbNup170 is elliptical in shape. Phylogenetic analysis based on the Nup170 and homologous sequences showed that N. bombycis clustered together with Vairimorpha ceranae and Vairimorpha apis. The immunofluorescence localization results showed that the NbNup170 was located on the plasma membrane of the dormant spore and transferred to the surface of sporoplasm in a punctate pattern when the dormant spore has finished germination, and that NbNup170 was distributed on the nuclear membrane and both sides of the nuclei of early proliferative phase, and only on the nuclear membrane during sporogonic phase in the N. bombycis. qPCR analysis showed that the relative expression level of NbNup170 maintained at a low level from 30 to 78 h post-infection with N. bombycis, then reached the highest at 102 h, while that of NbNup170 was repressed at a very low level throughout its life cycle by RNA interference. These results suggested that NbNup170 protein is involved in the proliferative phase and active during the sporogonic phase of N. bombycis. [ABSTRACT FROM AUTHOR]

Microsporidia are obligate single-celled eukaryote parasites. Microsporidian infection can cause large economic losses to beneficial insects such as silkworms and honey bees. Identification of resistance biomacromolecules and breeding of transgenic lines resistant to the microsporidian Nosema bombycis are important for disease management. We previously used transcriptome analysis to identify a guanylate binding protein family BmAtlastin-n gene that was significantly upregulated after Nosema bombycis infection, and we determined that the molecule was highly expressed in resistance-related tissues such as the midgut, fat body and the epidermis. The transgenic silkworm line overexpressing BmAtlastin-n biomolecules had economic characters similar to those of non-transgenic lines. The transgenic OE-BmAtlastin-n lines had significantly improved survival after microspore infection. We used RT-PCR and H&E staining to show that the number of spores in the transgenic lines was significantly lower than in the control lines. In this study, we identified a BmAtlastin-n macromolecule with resistance to N. bombycis and developed a transgenic line. The results improved understanding of the GBP protein family and provided biomacromolecule material for the treatment and prevention of microsporidia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Activation of cellular defence and immunity in the ovary is critical to protect it from pathogenic infection. In this study cellular and innate immunity in larval and pupal ovaries of Bombyx mori induced by the microsporidian Nosema bombycis is reported. Autophagic vacuoles enclosing spores in flat cells of the larval ovarian disc revealed cellular defence. In the follicular epithelium of differentiated previtellogenic ovarioles of infected pupae, a network of RER and phagosomes observed probably to maintain cellular homeostasis. In larval and pupal ovaries, paralytic peptide titre, tyrosine content and phenoloxidase activity were enhanced revealing greater melanisation, which was confirmed by activation of melanisation genes, DDC, prophenoloxidase activating enzyme, prophenoloxidase 1 (PPO1) and PPO2 after infection. βGRP-2, βGRP-4, CTL-11, Humoral lectin, Toll-6, Spätzle, Cactus, Relish, Pelle, and Dorsal genes showed differential expression in infected larval and pupal ovaries. Pupal ovarioles showed decreased expression and lower Spätzle titre than the larval ovary revealing suppression of innate immunity in the pupal ovary. Dorsal showed decreased expression, however, Pelle enhanced expression to maintain immunity through alternate components in differentiated ovarioles. Our data showed that coupled with cellular defence reactions, innate immune reactions and melanisation are activated in the developing ovary to defend against N. bombycis infection. [ABSTRACT FROM AUTHOR]

• Nosema bombycis detection by NALFS is 10–100× more sensitive than agarose gel electrophoresis. • The presence of ≥2% infected eggs can be detected by NALFS. • Primer LSU323 targeting N. bombycis LSU rDNA is specific for some insect microsporidia. Nosema bombycis, the pathogen of silkworm pébrine, causes enormous economic losses to sericulture. As such, quarantine of commercial silkworm eggs represents an important safeguard to the silkworm industry. Here, we established a user-friendly detection system based on a nucleic acid lateral flow strip (NAFLS) that combines polymerase chain reaction (PCR) and a colloidal gold strip. PCR primers were designed based on the sequence of LSU rDNA of N. bombycis and has favourable specificity for common microsporidian isolates in silkworms. The forward and reverse primers were labeled on the 5′ end with biotin and carboxyfluorescein (FAM), respectively. Genomic DNA was extracted from egg samples and was used as a template for PCR, followed by subsequent detection by NALFS. The detection limit of purified N. bombycis genomic DNA was 1 pg, 100× more sensitive than that of agarose gel electrophoresis (AGE). Furthermore, the sensitivity of detection of simulated "infected" silkworm eggs was 10–100× higher than that of AGE. NALFS detected infection in 27 of 29 samples of silkworm eggs oviposited by female moths infected in lab; ≥2% infected eggs per batch are detected as positive, while ≥40% infected eggs per batch are required for detection by AGE. Collectively, NALFS is easy to use and has great potential for widespread use in the detection of N. bombycis in silkworm egg production. [ABSTRACT FROM AUTHOR]

Abstract The centriole in eukaryotes functions as the cell's microtubule-organizing center (MTOC) to nucleate spindle assembly. The evolutionarily conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. Microsporidia possess the spindle plaque instead of centriole as their MTOC to nucleate spindle assembly. However, little is known about the components of spindle plaques in microsporidia. In our present study, we identified a SAS-6 protein in the microsporidium Nosema bombycis and named it as NSAS-6. The NSAS-6 gene contains a complete ORF of 1104 bp in length that encodes a 367-amino acid polypeptide. NSAS-6 consists of a conserved N-terminal domain and a coiled-coil domain. The high identity of SAS-6 homologous sequences from microsporidia indicates that SAS-6 is a conserved protein in microsporidia. Immunolocalization in sporoplasms, intracellular stages and mature spores showed that NSAS-6 probably localizes to the nucleus of N. bombycis and exists throughout the life cycle of N. bombycis. These results suggest that NSAS-6 is required in cell morphogenesis and division in N. bombycis. The function and structure of NSAS-6 should be the focus for further studies, which is essential to elucidate the role of SAS-6 in spindle plaque assembly. Graphical abstract Unlabelled Image Highlights • • NSAS-6 localizes to the nucleus of N. bombycis. • •During extrusion of sporoplasms from mature spores, NSAS-6 is released simultaneously. • • NSAS-6 exists throughout the life cycle of N. bombycis. • • NSAS-6 is ancestrally present in the Fungi/microsporidia but lost in many fungal groups. [ABSTRACT FROM AUTHOR]

The secretion of hexokinases (HKs) by microsporidia followed by their accumulation in insect host nuclei suggests that these enzymes play regulatory and catalytic roles in infected cells. To confirm whether HKs exert catalytic functions in insect cells, we expressed in E. coli the functionally active HKs of two entomopathogenic microsporidia, Nosema bombycis and Nosema ceranae, that cause silkworm and honey bee nosematoses. N. bombycis HK with C-terminal polyHis tag and N. ceranae enzyme with N-terminal polyHis tag were cloned into pOPE101 and pRSET vectors, respectively, and overexpressed. Specific activities of N. bombycis and N. ceranae enzymes isolated by metal chelate affinity chromatography were 29.2 ± 0.5 and 60.2 ± 1.2 U/mg protein at an optimal pH range of 8.5–9.5. The kinetic characteristics of the recombinant enzymes were similar to those of HKs from other parasitic and free-living organisms. N. bombycis HK demonstrated Km 0.07 ± 0.01 mM and kcat 1726 min−1 for glucose, and Km 0.39 ± 0.05 mM and kcat 1976 min−1 for ATP, at pH 8.8. N. ceranae HK showed Km 0.3 ± 0.04 mM and kcat 3293 min−1 for glucose, and Km 1.15 ± 0.11 mM and kcat 3732 min−1 for ATP, at the same pH value. These data demonstrate the capability of microsporidia-secreted HKs to phosphorylate glucose in infected cells, suggesting that they actively mediate the effects of the parasite on host metabolism. The present findings justify further study of the enzymes as targets to suppress the intracellular development of silkworm and honey bee pathogens. [ABSTRACT FROM AUTHOR]

Abstract Bmtutl-519 is an isoform of the Bombyx Turtle protein and a member of the immunoglobulin superfamily (IgSF). The relative expression level of Bmtutl-519 was significantly upregulated when BmN cells were infected by Nosema bombycis. The subcellular localization of Bmtutl-519 was studied using an indirect immunoinfluscent assay (IFA), Co-localization assay, Western blotting, and enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bmtutl-519 expression plasmid. The results indicate that Bmtutl-519 is distributed in both the cytoplasm and the cell membrane of BmN cells. Bmtutl-519 may be involved in the infection process of N. bombycis as a cell surface receptor or regulatory factor. Interaction analysis of Bmtutl-519 with NbSWP26, a spore wall protein of N. bombycis involved in host cell adherence and infection, showed that the C-terminal heparin-binding motif (HBM) of NbSWP26 mediates the interaction between these two proteins. Mutation of the NbSWP26 HBM at K208G, K209G, K210G, and K213G led to a loss of the ability to bind the Bmtutl-519 protein. Spore adherence and infection assays showed that Bmtutl-519 enhances the binding ability of N. bombycis to the host cell surface, but this did not enhance host cell infection by N. bombycis. In contrast, the sustained high expression of Bmtutl-519 in BmN cells inhibited the proliferation of N. bombycis spores. Graphical abstract Image 10356 Highlights • Bmtutl-519 was distributed in both cytoplasm and cell membrane of BmN cells. • The interaction between Bmtutl-519 and NbSWP26 was mediated by the C-terminal heparin-binding motif (HBM) of NbSWP26. • Bmtutl-519 may serve as a receptor to N. bombycis spores through recognition of NbSWP26. • Bmtutl-519 enhances the binding ability of N. bombycis to BmN cell, but the overexpression of Bmtutl-519 inhibited the proliferation of spores. [ABSTRACT FROM AUTHOR]

Abstract ATP-binding cassette (ABC) transporters comprise the largest family of transmembrane proteins and are found in all domains of life. The ABCs are involved in a variety of biological processes and as exporters play important roles in multidrug resistance. However, the ABC transporters have not been addressed in microsporidia, which are a very large group of obligate intracellular parasites that can infect nearly all animals, including humans. Here, a total of 234 ABC transporters were identified from 18 microsporidian genomes and classified into five subfamilies, including 74 ABCBs, 2 ABCCs, 18 ABCEs, 15 ABCFs, 102 ABCGs and 23 uncategorized members. Two subfamilies, ABCA and ABCD, are found in most organisms, but lost in microsporidia. Phylogenetic analysis indicated that microsporidian ABCB and ABCG subfamilies expanded by recent gene duplications, which resulted in the two largest subfamilies in microsporidia. Functional analysis via qRT-PCR and Western blotting revealed that NoboABCG1.1 , an ABCG member of Nosema bombycis , is expressed in mature spores and up-regulated from 1 dpi to 6 dpi in infected silkworm midgut. IFA and IEM analysis showed that NoboABCG1.1 is localized on the plasma membrane of the sporoplasm, meront and mature spore. The propagation of N. bombycis was significantly inhibited after the RNAi of NoboABCG1.1 expression, indicating that NoboABCG1.1 is important to the pathogen proliferation. In conclusion, our study uncovered that the ABCs evolved during microsporidia adaption to intracellular parasitism and play important roles in the pathogen development. Highlights • 234 ABC genes are identified from 18 microsporidian genomes • Microsporidian ABC genes are assigned to five subfamilies: ABCB, ABCC, ABCE, ABCF, ABCG • Gene expansions of ABCB and ABCG subfamily were caused by recent gene duplications • NoboABCG1.1 is localized on the plasma membrane of sporoplasm, meront and mature spore in Nosema bombycis. • NoboABCG1.1 plays important role in substrate transportation during spore propagation. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Co-infections with PhopGV and a microsporidium was observed in Phthorimaea operculella larvae. • Mortality of P. operculella caused by PhopGV was reduced in microsporidian infected larvae. • The microsporidium was morphologically and genetically identified as Nosema bombycis. Abstract An antagonistic effect of a microsporidium (Nosema sp.) infection on the virulence of Phthorimaea operculella granulovirus (PhopGV) was recorded in potato tuber moth (Phthorimaea operculella) larvae with mixed infections. When the P. operculella colony was infected at a high rate (42.8–100%) with the microsporidium, it was less susceptible to the isolate PhopGV-GR1.1. A virus concentration 1.89 × 105 higher was necessary to cause the same level of mortality produced in the P. operculella colony when it was uninfected or had a low level of infection with the microsporidium (0–30%). This antagonistic effect was driven by a Nosema isolate (termed Nosema sp. Phop) that was purified from microsporidian-infected P. operculella individuals. The purified microsporidium was characterised by morphological features, including size, filament coils and different developmental stages using transmission electron microscopy (TEM). On the molecular level, the partial cistron rDNA information of the small ribosomal subunit (SSU), internal transcribed spacer (ITS), and the large ribosomal subunit (LSU) were identified. Phylogenetic analyses revealed that the newly described microsporidium belongs to the "true Nosema " clade. Partial sequence information of the RNA polymerase II largest subunit (RPB1) suggested that Nosema bombycis is the closest relative (98% identity). The morphological and phylogenetic characteristics suggest that it is an isolate of N. bombycis. Interactions of microsporidia and betabaculoviruses are rarely described in the literature, although mixed infections of different pathogens seem to be rather common events, ranging from antagonistic to mutualistic interactions. The observed antagonistic relationship between the Nosema sp. and PhopGV-GR1.1 showed that pathogen interactions need to be considered when single pathogens are applied to insect populations in the context of biological control of insect pests. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The portable and rapid detection of biomolecules via pH meters to monitor the concentration of hydrogen ions (H + ) from biological reactions (e.g. loop-mediated isothermal amplification, LAMP) has attracted research interest. However, this assay strategy suffered from inherent drawback of low sensitivity, resulting in great limitations in practical applications. Herein, a novel electrochemical biosensor was constructed for highly sensitive detection of Nosema bombycis gene DNA (PTP1) through transducing chemical stimuli H + from PTP1-based LAMP into electrochemical output signal of electroactive ferrocene (Fc). With use of target PTP1 as the template, the H + from LAMP induced the conformational switch of pH-responsive DNA nanostructures (DNA NSs, Fc-Sp@Ts) that was assembled by the hybridization of Fc-labeled signal probe (Fc-Sp) with DNA-based receptor (Ts). Due to the folding of Ts into stable triplex structure at decreased pH, the configuration change of Fc-Sp@Ts led to the releasing of Fc-Sp, which was subsequently immobilized in the electrode interface through the hybridization with the capture probe modified with -SH (SH-Cp), generating amplified electrochemical signal from Fc. The developed biosensor for PTP1 exhibited a reliable linear range of 1 fg µL −1 to 50 ng µL −1 with the limit of detection of 0.31 fg µL −1 . Thus, by the regulation of H + from LAMP reaction on DNA NSs allostery, this novel and simple transduction scheme would be interesting and promising to open up a novel analytical route for sensitive monitoring of different target DNAs in related disease diagnosis. [ABSTRACT FROM AUTHOR]

Microsporidia are highly specialized obligate intracellular, spore forming divergent fungi with a wide variety host range that includes most vertebrates and invertebrates. The resistant spores are surrounded by a rigid cell wall which consists of three layers: the electron-lucent chitin and protein inner endospore, the outer-electron-dense and mainly proteinaceous exospore and plasma membrane. Interestingly, microsporidia owns a special invasion organelle, called polar tube, coiled within the interior of the spore wall and attached to anchoring disk at the anterior end of spore. Spore wall and polar tube are the major apparatuses for mature spores adhering and infecting to the host cells. In this review, we summarize the research advances in spore wall proteins (SWPs) related to spore adherence and infection, and SWPs and deproteinated chitin spore coats (DCSCs) interaction associated with SWPs deposit processes and spore wall assembly. Furthermore, we highlight the SWPs-polar tube proteins (PTPs) interaction correlated to polar tube orderly orientation, arrangement and anchorage to anchoring disk. Based on results obtained, it is helpful to improve understanding of the spore wall assembly and polar tube orderly arrangement mechanisms and molecular pathogenesis of microsporidia infection. Also, such information will provide a basis for developing effective control strategies against microporidia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nosema bombycis is a destructive, obligate intracellular parasite of the Bombyx mori. In this study, a single-chain variable fragment (scFv) dependent technology is developed for the purpose of inhibiting parasite proliferation in insect cells. The scFv-G4, which we prepared from a mouse G4 monoclonal antibody, can target the N. bombycis spore wall protein 12 (NbSWP12). Indirect immunofluorescence assays showed that NbSWP12 located mainly on the outside of the N. bombycis cytoskeleton, although some of it co-localized with β-tubulin in the meront-stage of parasites. When meront division began, NbSWP12 became concentrated at both ends of each meront. Western blotting showed that scFv-G4 could express in Sf9-III cells and recognized native NbSWP12. The transgenic Sf9-III cell line showed better resistance than the controls when challenged with N. bombycis, indicating that NbSWP12 is a promising target in this parasite and this scFv dependent strategy could be a solution for construction of N. bombycis-resistant Bombyx mori. [ABSTRACT FROM AUTHOR]

The silkworm (Bombyx mori) is a model species of lepidopteran insect. Microsporidium spp. are obligate intracellular eukaryotic parasites. Infection by the microsporidian Nosema bombycis (Nb) results in an outbreak of Pébrine disease in silkworms and causes substantial losses to the sericulture industry. It has been suggested that Nb depends on nutrients from host cells for spore growth. However, little is known about changes in lipid levels after Nb infection. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was performed to analyze the effect of Nb infection on lipid metabolism in the midgut of silkworms. A total of 1601 individual lipid molecules were detected in the midgut of silkworms, of which 15 were significantly decreased after Nb challenge. Classification, chain length, and chain saturation analysis revealed that these 15 differential lipids can be classified into different lipid subclasses, of which 13 belong to glycerol phospholipid lipids and two belong to glyceride esters. The results indicated that Nb uses the host lipids to complete its own replication, and the acquisition of host lipid subclasses is selective; not all lipid subclasses are required for microsporidium growth or proliferation. Based on lipid metabolism data, phosphatidylcholine (PC) was found to be an important nutrient for Nb replication. Diet supplementation with lecithin substantially promoted the replication of Nb. Knockdown and overexpression of the key enzyme phosphatidate phosphatase (PAP) and phosphatidylcholine (Bbc) for PC synthesis also confirmed that PC is necessary for Nb replication. Our results showed that most lipids in the host midgut decreased when silkworms were infected with Nb. Reduction of or supplementation with PC may be a strategy to suppress or promote microsporidial replication. • Infection of Nosema bombycis alters the lipid metabolism of its host silkworm. • Nosema bombycis depends on host's lipids for effective proliferation. • Lecithin (phosphatidylcholine; PC) acts as host metabolite for the proliferation of Nb in silkworm. [ABSTRACT FROM AUTHOR]

Several species of bumblebees have recently experienced range contractions and possible extinctions. While threats to bees are numerous, few analyses have attempted to understand the relative importance of multiple stressors. Such analyses are critical for prioritizing conservation strategies. Here, we describe a landscape analysis of factors predicted to cause bumblebee declines in the USA. We quantified 24 habitat, land-use and pesticide usage variables across 284 sampling locations, assessing which variables predicted pathogen prevalence and range contractions via machine learning model selection techniques. We found that greater usage of the fungicide chlorothalonil was the best predictor of pathogen (Nosema bombi) prevalence in four declining species of bumblebees. Nosema bombi has previously been found in greater prevalence in some declining US bumblebee species compared to stable species. Greater usage of total fungicides was the strongest predictor of range contractions in declining species, with bumblebees in the northern USA experiencing greater likelihood of loss from previously occupied areas. These results extend several recent laboratory and semifield studies that have found surprising links between fungicide exposure and bee health. Specifically, our data suggest landscape-scale connections between fungicide usage, pathogen prevalence and declines of threatened and endangered bumblebees. [ABSTRACT FROM AUTHOR]

Nosema bombycis is an obligate intracellular parasite, which can cause pébrine disease. To investigate the effects of N. bombycis infection, 5th-instar silkworms were challenged with N. bombycis isolate CQ1, and two-dimensional gel electrophoresis analysis was performed to analyze the differentially expressed proteins in infected and uninfected silkworm fat bodies 1, 2, 4, 6 and 8 days post-infection (dpi). 46 differentially expressed proteins were identified at the 5 time points using MALDI-TOF/TOF MS. The changed proteins mainly involved in immune response, energy metabolism, and molecular synthesis. Overall, the identified proteins may provide important insights into the mechanisms of the silkworm response to N. bombycis infection. [ABSTRACT FROM AUTHOR]

Nosema bombycis is the pathogen of pébrine which brings heavy losses to sericulture every year. As a member of serine proteases, subtilisin-like protease (SLP) is related to the pathogenicity in fungi. In this study, we characterized a novel 63.8 kDa subtilisin-like protease NbSLP2 with a predicted transmembrane domain from Microsporidia, N. bombycis . RT-PCR showed that the transcript of NbSLP2 was detected from third day post infection. Immunofluorescence assay (IFA) indicated that NbSLP2 mainly scattered around the spore wall of N. bombycis . Co-immunoprecipitation data and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis revealed that NbSLP2 directly interacts with septin2 of N. bombycis , which is a cytoskeletal protein. IFA showed that NbSLP2 and Nbseptin2 co-localized beneath the spore wall. NbSLP2 can be pulled down by Nbseptin2, further confirming the interaction between NbSLP2 and Nbseptin2. As an important serine protease with a transmembrane domain, NbSLP2 interacting with Nbseptin2, a scaffold protein adjacent to the membrane may provide advantages to stabilize the NbSLP2 for its hydrolysis function. [ABSTRACT FROM AUTHOR]

As a common feature of eukaryotic proteins, tandem amino acid repeat has been studied extensively in both animal and plant proteins. Here, a comparative analysis focusing on the proteins having tandem repeats was conducted in eight microsporidia, including four mammal-infecting microsporidia ( Encephalitozoon cuniculi, Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon bieneusi) and four insect-infecting microsporidia ( Nosema apis, Nosema ceranae, Vavraia culicis and Nosema bombycis). We found that the proteins with tandem repeats were abundant in these species. The quantity of these proteins in insect-infecting microsporidia was larger than that of mammal-infecting microsporidia. Additionally, the hydrophilic residues were overrepresented in the tandem repeats of these eight microsporidian proteins and the amino acids residues in these tandem repeat sequences tend to be encoded by GC-rich codons. The tandem repeat position within proteins of insect-infecting microsporidia was randomly distributed, whereas the tandem repeats within proteins of mammal-infecting microsporidia rarely tend to be present in the N terminal regions, when compared with those present in the C terminal and middle regions. Finally, a hypothetical protein EOB14572 possessing four tandem repeats was successfully characterized as a novel endospore wall protein, which colocalized with polar tube of N. bombycis. Our study provided useful insight for the study of the proteins with tandem repeats in N. bombycis, but also further enriched the spore wall components of this obligate unicellular eukaryotic parasite. [ABSTRACT FROM AUTHOR]

In this report, we describe the cloning and characterization of a member of the immunoglobulin superfamily (IgSF); i.e., Turtle. The cDNA of Turtle was cloned from the silkworm Bombyx mori using the rapid amplification of cDNA ends (RACE) technique. Three isoforms of Bombyx Turtle were obtained, including Bmtutl-464, Bmtutl-519, and Bmtutl-810. The three isoforms had identical 27-amino acid signal peptides and four extracellular immunoglobulin (Ig) domains (IgI–IgIV). Sequence similarity and phylogenic analysis indicated that Bmtutl-810 belongs to the group of insect Turtle isoforms and shares 76.2% identity with Drosophila Turtle. Quantitative real-time PCR analysis revealed that the Bombyx Turtle isoforms were expressed throughout the entire development period, the highest levels of expression of Bmtutl-464 and Bmtutl-519 were observed at the second instar larvae stage, whereas that of Bmtutl-810 peaked at the embryonic stage. The ubiquitous expression of Bmtutl-464 , Bmtutl-519 , and Bmtutl-810 were observed in all studied tissues, except for Bmtutl-519 in the silk gland. The expression level of Bmtutl-464 was highest in the ovary, whereas that of Bmtutl-519 and Bmtutl-810 was highest in the hemolymph. Bmtutl-519 was upregulated in BmN cells infected by Nosema bombycis , We speculated that Bombyx Turtle was not only involved in neural development in silkworm, as well as Drosophila Turtle, but was also involved in the regulation of other biological functions. For example, Bmtutl-519 might be involved in N. bombycis infection and may play an important role in the immune response of silkworms to N. bombycis infection. [ABSTRACT FROM AUTHOR]

Microsporidia are a diverse group of eukaryotic organisms, capable of causing parasitic infections in both vertebrates and invertebrates. During the germination process, there is an increase in the osmotic pressure of microsporidian spores. As part of this study, we cloned a homologous aquaporin gene in Nosema bombycis, and named it Nosema bombycis aquaporin (NbAQP). Sequence analysis revealed that the NbAQP contains an open reading frame with a length of 750 bp and encodes a polypeptide of 249 amino acids. Amino acid sequence homology was greater than 50% that of five aquaporins from other microsporidian species. Indirect immunofluorescence (IFA) and immunogold electron microscopy showed NbAQP to be located predominantly in the spore wall of N. bombycis spores. The results of qRT-PCR analysis revealed that NbAQP expression remained high 0 h after inoculation and decreased sharply to 24 h, increased gradually from 2 days and peaked at 6 days. After expression of NbAQP in Xenopus laevis oocytes, it was observed that NbAQP can promote rapid penetration of water into oocytes. The associated permeation rate was 2–3 times that of the water-injected and uninjected oocytes. Antibody blocking experiments showed that the inhibition rate of spore germination was approximately 28% after antibody blocking. The difference in germination rate between the control group and the NbAQP group was significant (P < 0.05). This study shows for the first time that N. bombycis contains functional water channel proteins and provides a platform suitable for further research into the mechanisms underlying the regulation of NbAQP protein expression. Further study of NbAQP and their inhibitors may have significance for prevention of microsporidiosis. [ABSTRACT FROM AUTHOR]

In this study, an electrochemical proximity assay (ECPA)-coupled highly nonenzymatic amplifying strategy with electron mediator collected by a host-guest interaction was proposed for total protein of N. bombycis (TP N.b) detection. Antibody-dsDNA-gold nanoparticles functional Fe 3 O 4 probe (termed as Ab-dsDNA-Au@Fe 3 O 4 NPs), in which the Ab-dsDNA composed by adenine rich ssDNA (A 1 ) and Ab labeled capture ssDNA 1 (Ab-S 1 ), was first incubated with target TP N.b and another Ab labeled capture ssDNA 2 (Ab-S 2 ) to perform the proximity immunoreaction. Satisfyingly, the immunoreaction could not only produce sandwich immunocomplex with the proximity hybridization of S 1 and S 2 , but also exposed the trigger ssDNA A 1 to in situ form substantial methylene blue (MB) intercalated DNA concatamers via hybrid chain reaction (HCR). To further realize the nonenzymatic amplification, the MB intercalated in DNA concatemers was released into solution by duplex specific nuclease (DSN) and subsequently collected by prepared cucurbit(7)-uril (CB[7])/Au@Fe 3 O 4 NPs/GCE via host-guest interaction, which thus produced a significantly amplified signal for quantitative detection of target. Since the catalyst Fe 3 O 4 NPs on electrode surface could directly electrocatalyze the reduction of coimmobilized MB, the proposed system possessed superior advantages compared with that of traditionally enzymatic systems, which not only simplified the operation process, decreased measurement error but also improved catalytic efficiency and stability. [ABSTRACT FROM AUTHOR]

Microsporidia are eukaryotic, unicellular parasites that have been studied for more than 150 years. These organisms are extraordinary in their ability to invade a wide range of hosts including vertebrates and invertebrates, such as human and commercially important animals. A lack of appropriate labeling methods has limited the research of the cell cycle and protein locations in intracellular stages. In this report, an easy fluorescent labeling method has been developed to mark the proliferative and sporogonic phases of microsporidia Nosema bombycis in host cells. Based on the presence of chitin, Calcofluor White M2R was used to label the sporogonic phase, while β-tubulin antibody coupled with fluorescence secondary antibody were used to label the proliferative phase by immunofluorescence. This method is simple, efficient and can be used on both infected cells and tissue slices, providing a great potential application in microsporidia research. [ABSTRACT FROM AUTHOR]

The microsporidia Nosema bombycis is the insect pathogen of pebrine disease severely destructive to sericulture production. Here, we describe the use of Escherichia coli HT115 strain ( DE3) to express double-strand RNAs targeting the gene encoding ADP/ ATP protein in N. bombycis. The results showed that ds RNAs deferentially suppressed the gene expression during N. bombycis infection in the silkworm, and the effect waned gradually. Our results, for the first time, provide a tool to utilize the ds RNA expressed by recombinant E. coli to control the pebrine disease of the domestic silkworm. [ABSTRACT FROM AUTHOR]

Nosema bombycis, a microsporidium, is a pathogen of pebrine disease of silkworms, and its genomic DNA sequences had been determined. Thus far, the research of gene functions of microsporidium including N. bombycis cannot be performed with gain/loss of function. In the present study, we targeted to construct transgenic N. bombycis. Therefore, hemocytes of the infected silkworm were transfected with a non-transposon vector pIZT/V5-His vector in vivo, and the blood, in which the hemocyte with green fluorescence could be observed, was added to the cultured BmN cells. Furthermore, normal BmN cells were infected with germinated N. bombycis, and the infected cells were transfected with pIZT/V5-His. Continuous fluorescence observations exposed that there were N. bombycis with green fluorescence in some N. bombycis-infected cells, and the extracted genome from the purified N. bombycis spore was used as templates. PCR amplification was carried out with a pair of primers for specifically amplifying the green fluorescence protein (GFP) gene; a specific product representing the gfp gene could be amplified. Expression of the GFP protein through Western blotting also demonstrated that the gfp gene was perfectly inserted into the genome of N. bombysis. These results illustrated that exogenous gene can be integrated into N. bombycis genome by mediating with a non-transposon vector. Our research not only offers a strategy for research on gene function of N. bombycis but also provides an important reference for constructing genetically modified microsporidium utilized for biocontrol of pests. [ABSTRACT FROM AUTHOR]

A simple electrochemical proximity immunoassay (ECPA) system for the total protein of Nosema bombycis (TP N.b) detection has been developed on the basis of a new amplification strategy combined with target-induced proximity hybridization. The desirable ECPA system was achieved through following process: firstly, the methylene blue (MB) labeled hairpin DNA (MB-DNA) were immobilized on electrode through Au-S bonding. Then, the antibody labeled complementary single-stranded DNA probe (Ab 1 -S 1 ) hybridized with MB-DNA to open its hairpin structure, which led to the labeled MB far away from electrode surface. After that, the presence of target biomarker (TP N.b) and antibody labeled single-stranded DNA (Ab 2 -S 2 ) triggered the typical sandwich reaction and proximity hybridization, which resulted in the dissociation of Ab 1 -S 1 from electrode and the transformation of the MB-DNA into a hairpin structure with MB approaching to electrode surface. In consequence, the hairpin-closed MB was electrocatalyzed by the modified magnetic nanoparticles (Fe 3 O 4 NPs), leading to an increased and amplified electrochemical signal for the quantitative detection of TP N.b. In the present work, Fe 3 O 4 NPs were acted as catalyst to electrocatalyze the reduction of electron mediator MB for signal amplification, which could not only overcome the drawbacks of protein enzyme in electrocatalytic signal amplification but also shorten the interaction distance between catalyst and substance. Under optimal condition, the proposed ECPA system exhibited a wide linear range from 0.001 ng mL −1 to 100 ng mL − 1 with a detection limit (LOD) of 0.54 pg mL −1 . Considering the desirable sensitivity and specificity, as well as the novel and simple features, this signal amplified ECPA system opened an opportunity for quantitative analysis of many other kinds of protein biomarker. [ABSTRACT FROM AUTHOR]

Emergent fungal diseases are critical factors in global biodiversity declines. The fungal pathogen Nosema bombi was recently found to be widespread in declining species of North American bumble bees (Bombus), with circumstantial evidence suggesting an exotic introduction from Europe. This interpretation has been hampered by a lack of knowledge of global genetic variation, geographic origin, and changing prevalence patterns of N. bombi in declining North American populations. Thus, the temporal and spatial emergence of N. bombi and its potential role in bumble bee decline remain speculative. We analyze Nosema prevalence and genetic variation in the United States and Europe from 1980, before an alleged introduction in the early 1990s, to 2011, extracting Nosema DNA from Bombus natural history collection specimens from across this time period. Nosema bombi prevalence increased significantly from low detectable frequency in the 1980s to significantly higher frequency in the mid- to late-1990s, corresponding to a period of reported massive infectious outbreak of N. bombi in commercial bumble bee rearing stocks in North America. Despite the increased frequency, we find no conclusive evidence of an exotic N. bombi origin based on genetic analysis of global Nosema populations; the widespread Nosema strain found currently in declining United States bumble bees was present in the United States before commercial colony trade. Notably, the US N. bombi is not detectably different from that found predominantly throughout Western Europe, with both regions characterized by low genetic diversity compared with high levels of diversity found in Asia, where commercial bee breeding activities are low or nonexistent. [ABSTRACT FROM AUTHOR]

[Display omitted] • The main function of Sec61 complex is participating in the transport of polypeptide chains across endoplasmic reticulum. • NbSec61α and NbSec61γ were mainly distributed in the perinuclear region of N. bombycis in different developmental phases. • Knockdown of NbSec61α suppressed significantly the expression level of Nb Sec61γ and NbssrRNA genes. The main function of Sec61 complex is participating in the transport of polypeptide chains across the endoplasmic reticulum. The Sec61α subunit is the largest subunit of the Sec61 complex and shows high degree of conservation. In this study, we identified the NbSec61α and NbSec61γ genes in the microsporidian Nosema bombycis for the first time. Multiple sequence alignment showed that the sequence similarity between NbSec61α and homologous proteins of other microsporidia was greater than 48 %. NbSec61α contains a "plug" domain (amino acids 40–74) unique to the Sec61/SecY complex. Phylogenetic analysis based on NbSec61α and NbSec61γ indicated that the N. bombycis was closely related to Nosema granulosis , Nosema ceranae and Nosema apis. Indirect immunfluorescence assay showed that NbSec61α and NbSec61γ were mainly distributed in the perinuclear region of N. bombycis in different developmental phases. qRT-PCR results revealed that the expression level of NbSec61α gene increased in the early stage and reached the highest at 48 h, then decreased in the late stages. After knockdown of NbSec61α , the expression of NbSec61α , NbSec61γ and NbssrRNA genes were all significantly down-regulated. These results suggest that the NbSec61α and NbSec61γ may play an important role in the intracellular development of N. bombycis. [ABSTRACT FROM AUTHOR]

[Display omitted] • IAP was identified in the microsporidian Nosema bombycis for the first time. • NbIAP may be not participate in the regulation of the host cell apoptosis. • NbIAP gene silencing directly inhibits the proliferation of N. bombycis. Many organisms go through a process of programmed cell death called apoptosis while newer cells are created. This has the effect of protecting the organism from cellular parasites and is a major line of defense against invading organisms. Apoptosis inhibitors, then, play an important role in aiding infectious agents by inhibiting caspase protease and thus the apoptopic pathway. In this study, we identified an inhibitor of apoptosis protein (IAP) in the microsporidian Nosema bombycis (NbIAP). NbIAP a composed of 218 amino acids containing two overlapping domains; the BIR domain and a zf-C3HC domain. We show, through indirect immunofluorescence, that NbIAP is present throughout the life cycle of N. bombycis and is localized in the nucleus of the parasite and therefor does not act on caspase protease directly. qRT-PCR analysis shows that the expression of the NbIAP gene was the highest on the first day of infection, then decreased to a relatively stable level. In addition, we show that the downregulation of the NbIAP gene directly inhibits the proliferation of N. bombycis. These findings suggest that NbIAP plays an important role in the N. bombycis life cycle. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]