The microsporidian Nosema bombycis is an obligate intracellular parasite of Bombyx mori, that lost its intact tricarboxylic acid cycle and mitochondria during evolution but retained its intact glycolysis pathway. N. bombycis hexokinase (NbHK) is not only a rate-limiting enzyme of glycolysis but also a secretory protein. Indirect immunofluorescence assays and recombinant HK overexpressed in BmN cells showed that NbHK localized in the nucleus and cytoplasm of host cell during the meront stage. When N. bombycis matured, NbHK tended to concentrate at the nuclei of host cells. Furthermore, the transcriptional profile of NbHK implied it functioned during N. bombycis’ proliferation stages. A knock-down of NbHK effectively suppressed the proliferation of N. bombycis indicating that NbHK is an important protein for parasite to control its host.

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.

As a silkworm pathogen, the microsporidian N. bombycis can be transovarially transmitted from parent to offspring and seriously impedes sericulture industry development. Previous studies found that Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are involved in regulating diverse cellular processes, such as detoxification, pigmentation, and odorant sensing. Our results showed that BmUGT10295 and BmUGT8453 genes were specifically induced in infected silkworms, but other BmUGTs were not. Tissue distribution analysis of the two BmUGTs showed that the transcriptions of the two BmUGTs were mainly activated in the midgut and Malpighian tubule of infected silkworms. Furthermore, there were significantly fewer microsporidia in over-expressed BmUGTs compared with the control, but there were significantly more microsporidia in RNA interference BmUGTs compared with the control. These findings indicate that the two BmUGTs were induced by N. bombycis and provided resistance to the 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.

No abstract

Microsporidia are a group of obligated intracellular parasites that can infect nearly all vertebrates and invertebrates, including humans and economic animals. Microsporidian Vairimorpha necatrix is a natural pathogen of multiple insects and can massively proliferate by making tumor-like xenoma in host tissue. However, little is known about the subcellular structures of this xenoma and the proliferation features of the pathogens inside. Here, we characterized the V. necatrix xenoma produced in muscle cells of silkworm midgut. In result, the whitish xenoma was initially observed on the 12th day post infection on the outer surface of the midgut and later became larger and numerous. The observation by scanning electronic microscopy showed that the xenoma is mostly elliptical and spindle with dense pathogen-containing protrusions and spores on the surface, which were likely shedding off the xenoma through exocytosis and could be an infection source of other tissues. Demonstrated with transmission electron microscopy and fluorescent staining, the xenoma was enveloped by a monolayer membrane, and full of vesicle structures, mitochondria, and endoplasmic reticulum around parasites in development, suggesting that high level of energy and nutrients were produced to support the massive proliferation of the parasites. Multiple hypertrophic nuclei were found in one single xenoma, indicating that the cyst was probably formed by fusion of multiple muscle cells. Observed by fluorescence in situ hybridization, pathogens in the xenoma were in merongony, sporogony, and octosporogony, and mature stages. And mature spores were pushed to the center while vegetative pathogens were in the surface layer of the xenoma. The V. necatrix meront usually contained two to three nuclei, and sporont contained two nuclei and was wrapped by a thick membrane with high electron density. The V. necatrix sporogony produces two types of spores, the ordinary dikaryotic spore and unicellular octospores, the latter of which were smaller in size and packed in a sporophorous vesicle. In summary, V. necatrix xenoma is a specialized cyst likely formed by fusion of multiple muscle cells and provides high concentration of energy and nutrients with increased number of mitochondria and endoplasmic reticulum for the massive proliferation of pathogens inside.

Background: Microsporidia, a group of obligate intracellular parasites that can infect humans and nearly all animals, have lost the pathways for de novo amino acid, lipid and nucleotide synthesis and instead evolved strategies to manipulate host metabolism and immunity. The endoplasmic reticulum (ER) is a vital organelle for producing and processing proteins and lipids and is often hijacked by intracellular pathogens. However, little is known about how microsporidia modulate host ER pathways. Herein, we identified a secreted protein of Encephalitozoon hellem, EhHNTP1, and characterized its subcellular localization and functions in host cells.Methods: A polyclonal antibody against EhHNTP1 was produced to verify the protein subcellular localization in E. hellem-infected cells using indirect immunofluorescence assay (IFA) and Western blotting. HEK293 cells were transfected with wild-type or mutant EhHNTP1 fused with HA-EGFP, and the impacts on pathogen proliferation, protein subcellular localization and sequence functions were assessed. RNA sequencing of EhHNTP1-transfected cells was conducted to identify differentially expressed genes (DEGs) and pathway responses by bioinformatics analysis mainly with R packages. The DEGs in the transfected cells were experimentally confirmed with RT-qPCR and Western blotting. The regulatory effects of candidate DEGs were analyzed via RNA interference and cell transfection, and the effects were determined with RT-qPCR and Western blotting.Results: EhHNTP1 is secreted into the host nucleus, and its translocation depends on a nuclear localization signal sequence (NLS) at the C-terminus from amino acids 239 to 250. Transfection and overexpression of EhHNTP1 in HEK293 cells significantly promoted pathogen proliferation. RNA-seq of the transfected cells showed that genes involved in ER-associated degradation (ERAD), a quality control mechanism that allows for the targeted degradation of proteins in the ER, were prominently upregulated. Upregulation of the ERAD genes PDIA4, HERP, HSPA5 and Derlin3 determined by RNA-seq data was verified using RT-qPCR and Western blotting. Protein ubiquitination in the transfected cells was then assayed and found to be markedly increased, confirming the activation of ERAD. PDIA4 knockdown with RNAi significantly suppressed the expression of HERP, indicating that PDIA4 is a vital ERAD component exploited by EhHNTP1. Moreover, EhHNTP1ΔHRD, a deletion mutant lacking the histidine-rich domain (HRD) in the C-terminus, predominantly suppressed the upregulation of ERAD genes, indicating that the HRD is essential for EhHNTP1 functions.Conclusion: This study is the first report on a microsporidian secretory protein that targets the host nucleus to upregulate the ERAD pathway and subsequently promote protein ubiquitination. Our work provides new insights into microsporidia-host interactions.

Membrane applications for the separation of surfactant-stabilized emulsions are often constrained by a de ciency in permeability and anti-fouling properties. Herein, special wetted cotton fabric with a protective layer (P-MH@CF) for durable anti-fouling performance was designed by a two-step method, which was related to interfacial ion migration technology and unilateral spraying treatment. In detail, the immobilization of magnesium hydroxide caused the formation of the rough micro/nano structure of the cotton fabric surface. The stearic acid acted as a protective layer, like a quilt, protecting the membrane from contamination. Permeability of water and separation performance of P-MH@CF membrane were investigated systematically. For emulsion stabilized by SDS (SDS/Oil/H2O), the separation ux driven by gravity was approximately 500 L m -2 h -1 , and all separation e ciencies were over 99.3 %. CTAB/Oil/H2O emulsion and the Tween-60/Oil/H2O emulsion also could be successfully separated with high separation e ciency and separation ux. During the whole separation process, the oil droplets surrounded by surfactants were di cult to demulsify and gathered physically on the surface of the fabric to form a "creamy layer", which could be cleaned off so that the P-MH@CF membrane was not contaminated. In addition, the P-MH@CF membrane exhibited robust reusability for separation, which was promising for the remediation of oily wastewater containing surfactants.