SilkMeta: a comprehensive platform for sharing and exploiting pan-genomic and multi-omic silkworm data.

The silkworm Bombyx mori is a domesticated insect that serves as an animal model for research and agriculture. The silkworm super-pan-genome dataset, which we published last year, is a unique resource for the study of global genomic diversity and phenotype-genotype association. Here we present SilkMeta (http://silkmeta.org.cn), a comprehensive database covering the available silkworm pan-genome and multi-omics data. The database contains 1082 short-read genomes, 546 long-read assembled genomes, 1168 transcriptomes, 294 phenotype characterizations (phenome), tens of millions of variations (variome), 7253 long non-coding RNAs (lncRNAs), 18 717 full length transcripts and a set of population statistics. We have compiled publications on functional genomics research and genetic stock deciphering (mutant map). A range of bioinformatics tools is also provided for data visualization and retrieval. The large batch of omics data and tools were integrated in twelve functional modules that provide useful strategies and data for comparative and functional genomics research. The interactive bioinformatics platform SilkMeta will benefit not only the silkworm but also the insect biology communities.

Microsporidian Nosema bombycis hijacks host vitellogenin and restructures ovariole cells for transovarial transmission.

Microsporidia are a group of obligate intracellular parasites that infect almost all animals, causing serious human diseases and major economic losses to the farming industry. Nosema bombycis is a typical microsporidium that infects multiple lepidopteran insects via fecal-oral and transovarial transmission (TOT); however, the underlying TOT processes and mechanisms remain unknown. Here, we characterized the TOT process and identified key factors enabling N. bombycis to invade the ovariole and oocyte of silkworm Bombyx mori. We found that the parasites commenced with TOT at the early pupal stage when ovarioles penetrated the ovary wall and were exposed to the hemolymph. Subsequently, the parasites in hemolymph and hemolymph cells firstly infiltrated the ovariole sheath, from where they invaded the oocyte via two routes: (I) infecting follicular cells, thereby penetrating oocytes after proliferation, and (II) infecting nurse cells, thus entering oocytes following replication. In follicle and nurse cells, the parasites restructured and built large vacuoles to deliver themselves into the oocyte. In the whole process, the parasites were coated with B. mori vitellogenin (BmVg) on their surfaces. To investigate the BmVg effects on TOT, we suppressed its expression and found a dramatic decrease of pathogen load in both ovarioles and eggs, suggesting that BmVg plays a crucial role in the TOT. Thereby, we identified the BmVg domains and parasite spore wall proteins (SWPs) mediating the interaction, and demonstrated that the von Willebrand domain (VWD) interacted with SWP12, SWP26 and SWP30, and the unknown function domain (DUF1943) bound with the SWP30. When disrupting these interactions, we found significant reductions of the pathogen load in both ovarioles and eggs, suggesting that the interplays between BmVg and SWPs were vital for the TOT. In conclusion, our study has elucidated key aspects about the microsporidian TOT and revealed the key factors for understanding the molecular mechanisms underlying this transmission.

Reversible dougong structured receptor-ligand recognition for building dynamic extracellular matrix mimics.

Dynamic biomaterials excel at recapitulating the reversible interlocking and remoldable structure of the extracellular matrix (ECM), particularly in manipulating cell behaviors and adapting to tissue morphogenesis. While strategies based on dynamic chemistries have been extensively studied for ECM-mimicking dynamic biomaterials, biocompatible molecular means with biogenicity are still rare. Here, we report a nature-derived strategy for fabrication of dynamic biointerface as well as a three-dimensional (3D) hydrogel structure based on reversible receptor-ligand interaction between the glycopeptide antibiotic vancomycin and dipeptide d-Ala-d-Ala. We demonstrate the reversible regulation of multiple cell types with the dynamic biointerface and successfully implement the dynamic hydrogel as a functional antibacterial 3D scaffold to treat tissue repair. In view of the biogenicity and high applicability, this nature-derived reversible molecular strategy will bring opportunities for malleable biomaterial design with great potential in biomedicine.

Transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae.

Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-rearing industry. Whole-transcriptome analyses have revealed non-coding RNAs and their regulatory networks in N. bombycis infected embryos and larvae. However, transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae remains unclear. Here, we simultaneously compared the transcriptomes of N. bombycis and its host B. mori embryos of 5-day and larvae of 1-, 5- and 10-day during congenital infection. For the transcriptome of N. bombycis, a comparison of parasite expression patterns between congenital-infected embryos and larva showed most genes related to parasite central carbon metabolism were down-regulated in larvae during infection, whereas the majority of genes involved in parasite proliferation and growth were up-regulated. Interestingly, a large number of distinct or shared differentially expressed genes (DEGs) were revealed by the Venn diagram and heat map, many of them were connected to infection related factors such as Ricin B lectin, spore wall protein, polar tube protein, and polysaccharide deacetylase. For the transcriptome of B. mori infected with N. bombycis, beyond numerous DEGs related to DNA replication and repair, mRNA surveillance pathway, RNA transport, protein biosynthesis, and proteolysis, with the progression of infection, a large number of DEGs related to immune and infection pathways, including phagocytosis, apoptosis, TNF, Toll-like receptor, NF-kappa B, Fc epsilon RI, and some diseases, were successively identified. In contrast, most genes associated with the insulin signaling pathway, 2-oxacarboxylic acid metabolism, amino acid biosynthesis, and lipid metabolisms were up-regulated in larvae compared to those in embryos. Furthermore, dozens of distinct and three shared DEGs that were involved in the epigenetic regulations, such as polycomb, histone-lysine-specific demethylases, and histone-lysine-N-methyltransferases, were identified via the Venn diagram and heat maps. Notably, many DEGs of host and parasite associated with lipid-related metabolisms were verified by RT-qPCR. Taken together, simultaneous transcriptomic analyses of both host and parasite genes lead to a better understanding of changes in the microsporidia proliferation and host responses in embryos and larvae in N. bombycis congenital infection.

Establishment of a TaqMan probe-based qPCR assay for detecting microsporidia Enterospora epinepheli in grouper.

Enterospora epinepheli is an intranuclear microsporidian parasite causing serious emaciative disease in hatchery-bred juvenile groupers (Epinephelus spp.). Rapid and sensitive detection is urgently needed as its chronic infection tends to cause emaciation as well as white faeces syndrome and results in fry mortality. This study established a TaqMan probe-based real-time quantitative PCR assays targeting the small subunit rRNA (SSU) gene of E. epinepheli. The relationship between the standard curve of cycle threshold (Ct) and the logarithmic starting quantity (SQ) was determined as Ct = -3.177 lg (SQ) + 38.397. The correlation coefficient (R2 ) was 0.999, and the amplification efficiency was 106.4%. The detection limit of the TaqMan probe-based qPCR assay was 1.0 × 101 copies/µL and that is 100 times sensitive than the traditional PCR method. There is no cross-reaction with other aquatic microsporidia such as Ecytonucleospora hepatopenaei, Nucleospora hippocampi, Potaspora sp., Ameson portunus. The intra-assay and inter-assay showed great repeatability and reproducibility. In addition, the test of clinical samples showed that this assay effectively detected E. epinepheli in the grouper's intestine tissue. The established TaqMan qPCR assays will be a valuable diagnostic tool for the epidemiological investigation as well as prevention and control of E. epinepheli.

Non-coding RNAs identification and regulatory networks in pathogen-host interaction in the microsporidia congenital infection.

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.

First identification and coinfection detection of Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp. and Giardia duodenalis in diarrheic pigs in Southwest China.

BACKGROUND: Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp., and Giardia duodenalis (G. intestinalis) are enteric pathogens that cause diarrhea in pigs. This study aimed to determine the prevalence of these enteric parasites and their coinfection with E. bieneusi in diarrheic pigs in Southwest China (Chongqing and Sichuan) using nested polymerase chain reaction (nPCR) based methods. RESULTS: A total of 514 fecal samples were collected from diarrheic pigs from 14 pig farms in Chongqing (five farms) and Sichuan (nine farms) Provinces. The prevalence of Encephalitozoon spp., Cryptosporidium spp. and G. duodenalis was 16.14% (83/514), 0% (0/514), and 8.95% (46/514), respectively. Nested PCR revealed 305 mono-infections of E. bieneusi, six of E. cuniculi, two of E. hellem, and nine of G. duodenalis and 106 concurrent infections of E. bieneusi with the other enteric pathogens. No infections of E. intestinalis and Cryptosporidium species were detected. The highest coinfection was detected between E. bieneusi and E. cuniculi (10.5%, 54/514), followed by E. bieneusi and G. duodenalis (5.8%, 30/514) and E. bieneusi and E. hellem (2.9%, 15/514). E. bieneusi was the most frequently detected enteric pathogen, followed by E. cuniculi, G. duodenalis and E. hellem. There was a significant age-related difference in the prevalence of E. cuniculi in fattening pigs (χ2 = 15.266, df = 3, P = 0.002) and G. duodenalis in suckling pigs (χ2 = 11.92, df = 3, P = 0.008) compared with the other age groups. Sequence analysis of the ITS region of Encephalitozoon species showed two genotypes (II and III) for E. cuniculi and one (TURK1B) for E. hellem. Only G. duodenalis assemblage A was identified in all nested PCR-positive samples. E. bieneusi was found more often than other enteric pathogens. CONCLUSIONS: This study showed that E. bieneusi, Encephalitozoon spp. [E. cuniculi and E. hellem] and G. duodenalis were common enteric parasites in diarrheic pigs in Chongqing and Sichuan Provinces. In case of both mono-infection and coinfection, E. bieneusi was the most common enteric pathogen in diarrheic pigs. Thus, it may be a significant cause of diarrhea in pigs. Precautions should be taken to prevent the spread of these enteric parasites.

Characterization and fine mapping analysis of a major stable QTL qKnps-4A for kernel number per spike in wheat.

KEY MESSAGE: A major stable QTL for kernel number per spike was narrowed down to a 2.19-Mb region containing two potential candidate genes, and its effects on yield-related traits were characterized. Kernel number per spike (KNPS) in wheat is a key yield component. Dissection and characterization of major stable quantitative trait loci (QTLs) for KNPS would be of considerable value for the genetic improvement of yield potential using molecular breeding technology. We had previously reported a major stable QTL controlling KNPS, qKnps-4A. In the current study, primary fine-mapping analysis, based on the primary mapping population, located qKnps-4A to an interval of approximately 6.8-Mb from 649.0 to 655.8 Mb on chromosome 4A refering to 'Kenong 9204' genome. Further fine-mapping analysis based on a secondary mapping population narrowed qKnps-4A to an approximately 2.19-Mb interval from 653.72 to 655.91 Mb. Transcriptome sequencing, gene function annotation analysis and homologous gene related reports showed that TraesKN4A01HG38570 and TraesKN4A01HG38590 were most likely to be candidate genes of qKnps-4A. Phenotypic analysis based on paired near-isogenic lines in the target region showed that qKnps-4A increased KNPS mainly by increasing the number of central florets per spike. We also evaluated the effects of qKnps-4A on other yield-related traits. Moreover, we dissected the QTL cluster of qKnps-4A and qTkw-4A and proved that the phenotypic effects were probably due to close linkage of two or more genes rather than pleiotropic effects of a single gene. This study provides molecular marker resource for wheat molecular breeding designed to improve yield potential, and lay the foundation for gene functional analysis of qKnps-4A.

Detection of the pathogenic fungus Cordyceps farinosa in the Thitarodes armoricanus soil-rearing environment based on nucleic acid targets.

Cordyceps farinosa, an entomopathogenic fungus, infects and leads to high mortality of Thitarodes armoricanus larvae, which die soon after the infection of C. farinose, usually before the colonization of Ophiocordyceps sinensis owing to competitive inhibition and fruiting body formation. Therefore, monitoring C. farinosa in the O. sinensis cultivation environment is critical for minimizing the C. farinosa infection-induced losses. In this study, we initially designed a PCR primer pair (Tar-1F/Tar-1R) through open reading frame prediction and homology comparison of the C. farinosa genome sequence. This primer pair can detect both C. farinosa and Samsoniella hepiali. To further distinguish, primers (ITS5-172/ITS4-95) were then designed to selectively amplify the large ribosomal subunit sequences in the C. farinosa genome. All these primers were applied in combination for detection of C. farinosa in soil samples. The sensitivity reached a detection limit of 1 × 106 spores/g soil. In addition, these primers can detect the presence of C. farinosa in dead T. armoricanus larval samples. This newly established rapid detection method provides important information for C. farinosa control during O. sinensis cultivation.

The development of single-chain antibody anchored on the BmE cell membrane to inhibit BmNPV infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) poses a significant threat to sericulture production, and traditional sanitation practices remain the main strategy for controlling BmNPV infection. Although RNAi targeting BmNPV genes engineered into transgenic silkworms has shown to be a promising approach in reducing viral infection, it cannot block viral entry into host cells. Therefore, there is an urgent need to develop new effective prevention and control measures. In this study, we screened a monoclonal antibody 6C5 that potently neutralizes BmNPV infection by clamping the internal fusion loop of the BmNPVglycoprotein64 (GP64). Furthermore, we cloned the VH and VL fragments of mAb-6C5 from the hybridoma cell, and the eukaryotic expression vector of scFv6C5 was constructed to anchor the antibody on the cell membrane. The GP64 fusion loop antibody-expressing cells exhibited a reduced capacity for BmNPV infection. The results from our study provide a novel BmNPV control strategy and lay the foundation for the future development of transgenic silkworms with improved antiviral efficacy.

Bioclickable and mussel adhesive peptide mimics for engineering vascular stent surfaces.

Thrombogenic reaction, aggressive smooth muscle cell (SMC) proliferation, and sluggish endothelial cell (EC) migration onto bioinert metal vascular stents make poststenting reendothelialization a dilemma. Here, we report an easy to perform, biomimetic surface engineering strategy for multiple functionalization of metal vascular stents. We first design and graft a clickable mussel-inspired peptide onto the stent surface via mussel-inspired adhesion. Then, two vasoactive moieties [i.e., the nitric-oxide (NO)-generating organoselenium (SeCA) and the endothelial progenitor cell (EPC)-targeting peptide (TPS)] are clicked onto the grafted surfaces via bioorthogonal conjugation. We optimize the blood and vascular cell compatibilities of the grafted surfaces through changing the SeCA/TPS feeding ratios. At the optimal ratio of 2:2, the surface-engineered stents demonstrate superior inhibition of thrombosis and SMC migration and proliferation, promotion of EPC recruitment, adhesion, and proliferation, as well as prevention of in-stent restenosis (ISR). Overall, our biomimetic surface engineering strategy represents a promising solution to address clinical complications of cardiovascular stents and other blood-contacting metal materials.

Characterization and Biological Activities of Melanin from the Medicinal Fungi Ophiocordyceps sinensis.

Melanin is a complex natural pigment that is widely present in fungi. The mushroom Ophiocordyceps sinensis has a variety of pharmacological effects. The active substances of O. sinensis have been extensively studied, but few studies have focused on the O. sinensis melanin. In this study, the production of melanin was increased by adding light or oxidative stress, namely, reactive oxygen species (ROS) or reactive nitrogen species (RNS), during liquid fermentation. Subsequently, the structure of the purified melanin was characterized using elemental analysis, ultraviolet-visible absorption spectrum, Fourier transform infrared (FTIR), electron paramagnetic resonance (EPR), and pyrolysis gas chromatography and mass spectrometry (Py-GCMS). Studies have shown that O. sinensis melanin is composed of C (50.59), H (6.18), O (33.90), N (8.19), and S (1.20), with maximum absorbance at 237 nm and typical melanin structures such as benzene, indole, and pyrrole. Additionally, the various biological activities of O. sinensis melanin have been discovered; it can chelate heavy metals and shows a strong ultraviolet-blocking ability. Moreover, O. sinensis melanin can reduce the levels of intracellular reactive oxygen species and counteract the oxidative damage of H2O2 to cells. These results can help us to develop applications of O. sinensis melanin in radiation resistance, heavy metal pollution remediation, and antioxidant use.

Molecularly Imprinted Nanomaterials with Stimuli Responsiveness for Applications in Biomedicine.

The review aims to summarize recent reports of stimuli-responsive nanomaterials based on molecularly imprinted polymers (MIPs) and discuss their applications in biomedicine. In the past few decades, MIPs have been proven to show widespread applications as new molecular recognition materials. The development of stimuli-responsive nanomaterials has successfully endowed MIPs with not only affinity properties comparable to those of natural antibodies but also the ability to respond to external stimuli (stimuli-responsive MIPs). In this review, we will discuss the synthesis of MIPs, the classification of stimuli-responsive MIP nanomaterials (MIP-NMs), their dynamic mechanisms, and their applications in biomedicine, including bioanalysis and diagnosis, biological imaging, drug delivery, disease intervention, and others. This review mainly focuses on studies of smart MIP-NMs with biomedical perspectives after 2015. We believe that this review will be helpful for the further exploration of stimuli-responsive MIP-NMs and contribute to expanding their practical applications especially in biomedicine in the near future.

Microsporidiosis in Humans.

Microsporidia are obligate intracellular pathogens identified ∼150 years ago as the cause of pébrine, an economically important infection in silkworms. There are about 220 genera and 1,700 species of microsporidia, which are classified based on their ultrastructural features, developmental cycle, host-parasite relationship, and molecular analysis. Phylogenetic analysis suggests that microsporidia are related to the fungi, being grouped with the Cryptomycota as a basal branch or sister group to the fungi. Microsporidia can be transmitted by food and water and are likely zoonotic, as they parasitize a wide range of invertebrate and vertebrate hosts. Infection in humans occurs in both immunocompetent and immunodeficient hosts, e.g., in patients with organ transplantation, patients with advanced human immunodeficiency virus (HIV) infection, and patients receiving immune modulatory therapy such as anti-tumor necrosis factor alpha antibody. Clusters of infections due to latent infection in transplanted organs have also been demonstrated. Gastrointestinal infection is the most common manifestation; however, microsporidia can infect virtually any organ system, and infection has resulted in keratitis, myositis, cholecystitis, sinusitis, and encephalitis. Both albendazole and fumagillin have efficacy for the treatment of various species of microsporidia; however, albendazole has limited efficacy for the treatment of Enterocytozoon bieneusi. In addition, immune restoration can lead to resolution of infection. While the prevalence rate of microsporidiosis in patients with AIDS has fallen in the United States, due to the widespread use of combination antiretroviral therapy (cART), infection continues to occur throughout the world and is still seen in the United States in the setting of cART if a low CD4 count persists.

Mussel-Inspired Ligand Clicking and Ion Coordination on 2D Black Phosphorus for Cancer Multimodal Imaging and Therapy.

As a promising 2D nanocarrier, the biggest challenge of bare black phosphorus nanosheets (BP NSs) lies in the inherent instability, while it can be improved by surface modification strategies to a great extent. Considering the existing infirm BP NSs surface modification strategies, A mussels-inspired strong adhesive biomimetic peptide with azide groups for surface modification to increase the stability of BP NSs is synthesized. The azide groups on the peptide can quickly and precisely bind to the targeting ligand through click chemistry, solving the problem of nonspecificity of secondary modification of other mussel-mimicking materials. Besides, a catechol-Gd3+ coordination network is further constructed for magnetic resonance imaging (MRI) and inducing intracellular endo/lysosome escape. The fabricated BP-DOX@Gd/(DOPA)4 -PEG-TL nanoplatform exhibits enhanced antitumor abilities through synergetic chemo/photothermal effects both in vitro and in vivo.

Expression of anti-NbHK single-chain antibody in fusion with NSlmb enhances the resistance to Nosema bombycis in Sf9-III cells.

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.

Genome-Wide Characterization and Comparative Genomic Analysis of the Serpin Gene Family in Microsporidian Nosema bombycis.

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.

Down-Regulation of Lipid Metabolism in the Hepatopancreas of Shrimp Litopenaeus vannamei upon Light and Heavy Infection of Enterocytozoon hepatopenaei: A Comparative Proteomic Study.

Enterocytozoon hepatopenaei (EHP) is the pathogen of hepatopancreatic microsporidiosis (HPM) in shrimp. The diseased shrimp Litopenaeus vannamei exhibits a slow growth syndrome, which causes severe economic losses. Herein, 4D label-free quantitative proteomics was employed to analyze the hepatopancreas of L. vannamei with a light (EHPptp2 < 103 copies/50 ng hpDNA, L group) and heavy (EHPptp2 > 104 copies/50 ng hpDNA, H group) load of EHP to better understand the pathogenesis of HPM. Exactly 786 (L group) and 1056 (H group) differentially expressed proteins (DEPs) versus the EHP-free (C group) control were mainly clustered to lipid metabolism, amino acid metabolism, and energy production processing. Compared with the L group, the H group exhibited down-regulation significantly in lipid metabolism, especially in the elongation and degradation of fatty acid, biosynthesis of unsaturated fatty acid, metabolism of α-linolenic acid, sphingolipid, and glycerolipid, as well as juvenile hormone (JH) degradation. Expression pattern analysis showed that the degree of infection was positively correlated with metabolic change. About 479 EHP proteins were detected in infected shrimps, including 95 predicted transporters. These findings suggest that EHP infection induced the consumption of storage lipids and the entire down-regulation of lipid metabolism and the coupling energy production, in addition to the hormone metabolism disorder. These were ultimately responsible for the stunted growth.

Septin homologs cooperating in the Proliferative Stage of Microsporidia Nosema bombycis.

The single-celled pathogen Nosema bombycis, that can infect silkworm Bombyx mori and other lepidoptera including Spodoptera, is the first identified Microsporidia which has diplokaryotic nuclei throughout the life cycle. Septin proteins can form highly ordered filaments, bundles or ring structures related to the cytokinesis in fungi. Here, three septin proteins (NbSeptin1, NbSeptin2 and NbSeptin3) from Nosema bombycis CQ I are described. These proteins, appear to be conserved within the phylum Microsporidia. NbSeptins transcripts were detected throughout the pathogen developmental cycle and were significantly enhanced from second days of infection, which lead to our hypothesis that NbSeptins play a role in merogony. Immunofluorescence assay (IFA) revealed a broad distribution of NbSeptins in meronts and partly co-localization of NbSeptins. Interestingly, in some of meronts, NbSeptin2 and NbSeptin3 showed localization between the nuclei of the diplokaryon. Yeast two-hybrid and co-immunoprecipitation analysis verified that NbSeptins can interact with each other. Our findings suggest that NbSeptins can cooperate in the proliferation stage of Nosema bombycis and contribute towards the understanding of the rols of septins in microsporidia development.

Microsporidia infection upregulates host energy metabolism but maintains ATP homeostasis.

Microsporidia are a group of obligate intracellular parasites which lack mitochondria and have highly reduced genomes. Therefore, they are unable to produce ATP via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Instead, they have evolved strategies to obtain and manipulate host metabolism to acquire nutrients. However, little is known about how microsporidia modulate host energy metabolisms. Here, we present the first targeted metabolomics study to investigate changes in host energy metabolism as a result of infection by a microsporidian. Metabolites of silkworm embryo cell (BmE) were measured 48 h post infection by Nosema bombycis. Thirty metabolites were detected, nine of which were upregulated and mainly involved in glycolysis (glucose 6-phosphate, fructose 1,6-bisphosphate) and the TCA cycle (succinate, α-ketoglutarate, cis-aconitate, isocitrate, citrate, fumarate). Pathway enrichment analysis suggested that the upregulated metabolites could promote the synthesization of nucleotides, fatty acids, and amino acids by the host. ATP concentration in host cells, however, was not significantly changed by the infection. This ATP homeostasis was also found in Encephalitozoon hellem infected mouse macrophage RAW264.7, human monocytic leukemia THP-1, human embryonic kidney 293, and human foreskin fibroblast cells. These findings suggest that microsporidia have evolved strategies to maintain levels of ATP in the host while stimulating metabolic pathways to provide additional nutrients for the parasite.