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The American pumpkinseed Lepomis gibbosus, was introduced to Europe more than one hundred years ago. Currently it is a common fish in European freshwaters but relatively few specific parasites infect this fish in this new habitat. In Europe only a single species, Myxobolus dechtiari seems to represent the American myxosporean fauna of centrarchid fishes. M. dechtiari was found in both Portugal and Hungary. This species forms plasmodia with elongated shape inside the cartilaginous rays of gill filaments. In the advanced stage of infection, after disruption of plasmodia, small groups of myxospores remain enclosed in the cartilaginous gill rays causing distortions in the filaments. Myxospores were ellipsoidal in frontal view and lemon-shape in sutural, length 12.5 ± 0.46 (12–13.4) μm, width 10 ± 0.37 (9.6–10.4) μm, and thickness 7.4 ± 0.37 (7–8) μm; the polar capsules were pyriform, equal in size, length 5.6 ± 0.21 (5.3–6) μm, width 3.2 ± 0.16 (3–3.6) μm; Seven to eight polar tube coils were arranged perpendicularly to the capsule length. There was a small, round, 0.4 ± 0.1 (0.3–05) (N = 50) intercapsular appendix in the spores. The small subunit ribosomal DNA (ssrDNA) of M. dechtiari differed from other myxozoans sequenced to date. Phylogenetic analysis of the ssrDNA gene sequence placed this species in a clade including actinospores and Myxobolus species: Raabeia type1, Triactinomyxon sp., and Myxobolus osburni infecting the same host fish. The focus of our study was to prove that the pumpkinseed, a fish originated from North-America introduced one of its myxosporean parasite to Europe. Emphasis was put on to demonstrate the unique feature of this parasite causing infection in the cartilaginous gill rays., Graphical abstract Image 1, Highlights • American pumpkinseed fish were introduced to Europe as an ornamental fish. • Relatively few specific parasites are reported from the pumpkinseed in Europe. • The myxosporean Myxobolus dechtiari described originally in Canada infects the introduced host both in Hungary and Portugal. • SsrDNA sequences of M. dechtiari from pumpkinseed fish is provided.

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.

A new microsporidian species,Globosporidium parameciigen. nov., sp. nov., fromParamecium primaureliais described on the basis of morphology, fine structure, and SSU rRNA gene sequence. This is the first case of microsporidiosis inParameciumreported so far. All observed stages of the life cycle are monokaryotic. The parasites develop in the cytoplasm, at least some part of the population in endoplasmic reticulum and its derivates. Meronts divide by binary fission. Sporogonial plasmodium divides by rosette-like budding. Early sporoblasts demonstrate a well-developed exospore forming blister-like structures. Spores with distinctive spherical shape are dimorphic in size (3.7 ± 0.2 and 1.9 ± 0.2μm). Both types of spores are characterized by a thin endospore, a short isofilar polar tube making one incomplete coil, a bipartite polaroplast, and a large posterior vacuole. Experimental infection was successful for 5 of 10 tested strains of theParamecium aureliaspecies complex. All susceptible strains belong to closely relatedP. primaureliaandP. pentaureliaspecies. Phylogenetic analysis placed the new species in the Clade 4 of Microsporidia and revealed its close relationship toEuplotespora binucleata(a microsporidium from the ciliateEuplotes woodruffi), toHelmichia lacustrisandMrazekia macrocyclopis, microsporidia from aquatic invertebrates.

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens that do not vitrify well, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and cellular specimens that distribute poorly across grid squares. Here we present a general approach we call the ‘Waffle Method’ which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation of the spores on the grid. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with both manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. Additionally, we describe applications of the Waffle Method which are currently being explored. We propose the Waffle Method as a way to achieve many of the advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.

Microsporidia are eukaryotic, obligate intracellular parasites related to fungi. The success of microsporidia invasion process is based on their original invasion organelle: the polar tube. This multiproteic structure is composed by different proteins families named PTPs (Polar Tube Proteins) Our study focused on Anncaliia algerae, a microsporidian species initially described in mosquitoes and which is also able to infect humans. We first identified 5 PTPs (PTP1 to PTP5) encoding genes in the genome of A. algerae. Polyclonal antibodies raised against AaPTP1 to AaPTP4 allowed to confirm their localization on the polar tube and to study their solubility properties. In an interesting way, AaPTP3 is present on a large part of the polar tube except at its extremity. In contrast, AaPTP4 is only localized at the end-terminal part of the polar tube. With the aim to identify new potential polar tube components, sequential sporal protein extractions and a protocol of polar tube purification were established. Mass spectrometry analysis allowed the identification of 2 new polar tube proteins, AaPTP3b and AaPTP6, the last one is also specifically localized at the extremity of the polar tube. These analyses allowed also to identify a new sporoplasm protein. Finally, three potential invasion mechanisms were observed for A. algerae. Invasion tests showed that the PTP4 protein and the host actin play a role in A. algerae invasion processes. Therefore, we proposed an invasion model which will have to be completed by the identification of cellular receptors involved in invasion., Les microsporidies sont des microorganismes eucaryotes, parasites intracellulaires obligatoires, apparentés aux champignons. Leur processus d’invasion repose sur un appareil d’invasion très spécialisé : le tube polaire. Cette structure multiprotéique est composée de plusieurs familles de protéines appelées PTPs (Polar Tube Proteins). Nos travaux ont porté sur la caractérisation de ces protéines et de leur rôle dans les processus d’invasion chez l’espèce Anncaliia algerae, parasite de moustiques et de l’Homme. Tout d’abord, les gènes codant 5 PTPs (PTP1 à PTP5) ont été identifiés dans le génome d’A. algerae. La production d’anticorps contre les protéines AaPTP1 à AaPTP4 a permis de préciser la localisation de ces PTPs au niveau du tube polaire et d’étudier leurs propriétés de solubilité. De manière intéressante, la protéine AaPTP3 est présente sur une large partie du tube excepté à son extrémité. A l’inverse, la protéine AaPTP4 n’est localisée que dans la partie terminale du tube polaire. Dans le but d’identifier de nouveaux composants du tube polaire, des approches d’extractions séquentielles de protéines sporales et de purification des tubes polaires ont été mises en place. Des analyses en spectrométrie de masse ont alors permis d’identifier deux nouvelles PTPs, nommées AaPTP3b et AaPTP6, cette dernière étant également localisée à l’extrémité du tube polaire dévaginé. Ces analyses ont également mis en évidence une nouvelle protéine spécifique des sporoplasmes. Enfin, trois mécanismes d’invasion potentiels ont pu être observés chez A. algerae. Des tests d’inhibition de l’invasion ont également permis de mettre en évidence le rôle de la protéine PTP4 et de l’actine de l’hôte dans les processus d’invasion d’A. algerae. Un modèle d’invasion a donc été proposé et devra être complété par l’identification des récepteurs cellulaires impliqués.

Made available in DSpace on 2022-04-28T17:23:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-15 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) In the present study, we describe a co-infection of two microsporidians within the abdominal muscle of the Amazon River prawnMacrobrachium amazonicum from Brazil, detected through light and transmission electron microscopy and histopathological analysis. Two spore types and respective early developmental stages (meronts, sporonts, and sporoblasts) appeared grouped in numerous adjacent clusters among the muscle fibers of the host. Spores were initially divided into two morphotypes, Sp. 1 and Sp. 2, based on differences in shape, size, and internal organization. The Sp. 1 spores are pyriform to ovoid (4.3 +/- 0.3 x 3.7 +/- 0.4 mu m in size) and resided in groups of eight within a sporophorous vesicle. Sp. 1 spores had bilayered walls and long tape-like external filaments with irregular morphology and size, forming a complex contiguous membranous system attached to the spore wall. The umbrella-like anchoring disc of the spores was in continuity with an anisofilar polar tube arranged in 7-8 (rarely 9) coils. The Sp. 2 spores were rod-like in shape (3.1 +/- 0.5 x 0.8 +/- 0.1 mu m in diameter) and the polar tube had 7-8 coils. Their merogonic and sporogonic stages occurred within the sarcoplasm of the muscle cells in close contact with the myofibrils. Based on ultrastructural organization of the sporogonic stages, the Sp. 1 and Sp. 2 morphotypes probably belong to the genera Inodosporus and Myospora, respectively. Univ Estadual Paulista UNESP, Programa Posgrad Ciencias Biol Zool, Inst Biociencias, BR-13506900 Rio Claro, SP, Brazil FIT MG, Fac Inovacao & Tecnol Minas Gerais, BR-37900060 Passos, MG, Brazil Univ Estadual Paulista UNESP, Lab Morfol Invertebrados LMI, Dept Biol, Fac Ciencias Agr & Ve Jaboticabal, BR-14884900 Jaboticabal, SP, Brazil Univ Estadual Paulista UNESP, Ctr Aquicultura CAUNESP, Fac Ciencias Agr & Ve Jaboticabal, BR-14884900 Jaboticabal, SP, Brazil Univ Porto, Inst Invest & Inovacao Saude I3S, P-4200135 Porto, Portugal Univ Porto ICBAS UP, Lab Cell Biol, Inst Biomed Sci, P-4050313 Porto, Portugal Univ Inst Hlth Sci TOXRUN, Toxicol Res Unit, CESPU, P-4585116 Gandra, Portugal Univ Porto, Lab Anim Pathol, Interdisciplinary Ctr Marine & Environm Res CIIMA, P-4450208 Matosinhos, Portugal Univ Estadual Paulista UNESP, Programa Posgrad Ciencias Biol Zool, Inst Biociencias, BR-13506900 Rio Claro, SP, Brazil Univ Estadual Paulista UNESP, Lab Morfol Invertebrados LMI, Dept Biol, Fac Ciencias Agr & Ve Jaboticabal, BR-14884900 Jaboticabal, SP, Brazil Univ Estadual Paulista UNESP, Ctr Aquicultura CAUNESP, Fac Ciencias Agr & Ve Jaboticabal, BR-14884900 Jaboticabal, SP, Brazil CAPES: 001 CAPES: 1989/2014 CAPES: 23038.004309/2014-5 FAPESP: JP 2005#04707-5 FAPESP: 2018/13685-5 CNPq: 309298/2020-2

This article provides an outline of the classification of the kingdom Fungi (including fossil fungi. i.e. dispersed spores, mycelia, sporophores, mycorrhizas). We treat 19 phyla of fungi. These are Aphelidiomycota, Ascomycota, Basidiobolomycota, Basidiomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Entorrhizomycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota. The placement of all fungal genera is provided at the class-, order- and family-level. The described number of species per genus is also given. Notes are provided of taxa for which recent changes or disagreements have been presented. Fungus-like taxa that were traditionally treated as fungi are also incorporated in this outline (i.e. Eumycetozoa, Dictyosteliomycetes, Ceratiomyxomycetes and Myxomycetes). Four new taxa are introduced: Amblyosporida ord. nov. Neopereziida ord. nov. and Ovavesiculida ord. nov. in Rozellomycota, and Protosporangiaceae fam. nov. in Dictyosteliomycetes. Two different classifications (in outline section and in discussion) are provided for Glomeromycota and Leotiomycetes based on recent studies. The phylogenetic reconstruction of a four-gene dataset (18S and 28S rRNA, RPB1, RPB2) of 433 taxa is presented, including all currently described orders of fungi., Nalin N. Wijayawardene thanks Mushroom Research Foundation and National Science Foundation of China (No. NSFC 31950410558) for financially supporting this project. Kevin D. Hyde acknowledges the Foreign Experts Bureau of Yunnan Province, Foreign Talents Program (2018; grant no. YNZ2018002), Thailand Research grants entitled Biodiversity, phylogeny and role of fungal endophytes on above parts of Rhizophora apiculata and Nypa fruticans (grant no: RSA5980068), the future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species (grant no: DBG6080013), Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (grant no: RDG6130001). H.T. Lumbsch thanks support by the Grainger Bioinformatics Center. E. Malosso is grateful to CAPES for financial support (grant no. 88881.062172/2014-01). B.T. Goto, G.A. Silva and K. Jobim, L.C. Maia acknowledges CNPq (Brazilian Scientific Council, grants no. 465.420/2014-1, 307.129/2015-2 and 408011/2016-5) and CAPES for support. The study was partially supported by the National Science Centre, Poland, under Grants No. 2015/17/D/NZ8/00778 and 2017/25/B/NZ8/00473 to Julia Pawłowska. The research of Martin Kukwa received support from the National Science Centre (NCN) in Poland (project no 2015/17/B/NZ8/02441). Alan J.L. Phillips acknowledges the support from UID/MULTI/04046/2019 Research Unit grant from FCT, Portugal to BioISI. H. Zhang is financially supported by the National Natural Science Foundation of China (Project ID: NSF 31500017). S. Boonmee would like to thank the Thailand Research Fund (Project No. TRG6180001). Dong-Qin Dai and Li-Zhou Tang would like to thank the National Natural Science Foundation of China (No. NSFC 31760013, NSFC 31260087, NSFC 31460561), the Scientific Research Foundation of Yunnan Provincial Department of Education (2017ZZX186) and the Thousand Talents Plan, Youth Project of Yunnan Provinces for support. R. Phookamsak, M. Doilom, D. N. Wanasinghe, S.C. Karunarathna and J.C. Xu express sincere appreciations to Key Research Program of Frontier Sciences of the Chinese Academy of Sciences (grant no. QYZDY-SSW-SMC014) for research financial support. R. Phookamsak thanks the Yunnan Provincial Department of Human Resources and Social Security (grant no. Y836181261), Chiang Mai University and National Science Foundation of China (NSFC) project code 31850410489 for research financial support. S.C. Kaunarathna thanks CAS President’s International Fellowship Initiative (PIFI) for funding his postdoctoral research (No. 2018PC0006) and the National Science Foundation of China (NSFC) for funding this work under the project code 31851110759. S. Tibpromma would like to thank the International Postdoctoral Exchange Fellowship Program (number Y9180822S1), CAS President’s International Fellowship Initiative (PIFI) (number 2020PC0009), China Postdoctoral Science Foundation and the Yunnan Human Resources, and Social Security Department Foundation for funding her postdoctoral research. Yuri S. Tokarev, Elena S. Nassonova and Irma V. Issi are indebtful to Yuliya Y. Sokolova (Institute of Cytology RAS, St. Petersbug, Russia) and Anastasia V. Simakova (Tomsk State University, Tomsk, Russia) for kind permission of reproduction of electron microscopy images of Metchnikovella incurvata and Crepidulospora beklemishevi, respectively. Yuri S. Tokarev and Irma V. Issi thank Russian Foundation of Basic Research, grant number 17-04-00871 (taxonomy of Rozellomycota). Elena S. Nassonova thank Russian Foundation of Basic Research, grant number 18-04-01359 (early evolution of Microsporidia, phylogeny of Metchnikovellida). Adam Flakus and Pamela Rodriguez-Flakus are greatly indebted to all staff of the Herbario Nacional de Bolivia, Instituto de Ecología, Universidad Mayor de San Andrés, La Paz and the SERNAP (http://sernap.gob.bo), for their generous cooperation providing permits, assistance and facilities support for scientific studies. The research of AF and PRF were financially supported by the National Science Centre (NCN) in Poland (DEC-2013/11/D/NZ8/03274). Adam Flakus and Pamela Rodriguez-Flakus received additional support under statutory funds from the W. Szafer Institute of Botany, Polish Academy of Sciences, Krakow, Poland. The authors would like to thank Yunnan Innovation Platform for Development and Utilization of Symbiotic Fungi Resources for finance support. Li-Fang Zhang would like to thank grant-in-aid from Science and Technology Department of Yunnan Province (2018FD080) for finance support. Chun-Ying Deng thanks the Biodiversity Survey and Assessment Project of the Ministry of Ecology and Environment, China (2019HJ2096001006). Yingqian Kang would like to thank Guizhou Scientific Plan Project [(2019) 2873]; Excellent Youth Talent Training Project of Guizhou Province [(2017) 5639]; Guiyang Science and Technology Project [(2017) No. 5-19]; Talent Base Project of Guizhou Province, China [FCJD2018-22]; Research Fund of Education Bureau of Guizhou Province, China [(2018) 481]. D. N. Wanasinghe would like to thank the CAS President’s International Fellowship Initiative (PIFI) for funding his postdoctoral research (number 2019PC0008), the National Science Foundation of China and the Chinese Academy of Sciences for financial support under the following grants: 41761144055, 41771063 and Y4ZK111B01. Yuri K. Novozhilov and Oleg N. Shchepin acknowledge support from the Russian Foundation of Basic Research, project 18-04-01232 А. Ivana Kušan, Neven Matočec, Armin Mešić and Zdenko Tkalčec are grateful to Croatian Science Foundation for their financial support under the project grant HRZZ-IP-2018-01-1736 (ForFungiDNA). K. Tanaka would like to thank the Japan Society for the Promotion of Science (JSPS 19K06802)

All Microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore, transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200nm, noticeably larger than fixed-sectioned, or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5–6nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.

Microsporidia are a large group of fungus-related obligate intracellular parasites. Though many microsporidia species have been identified over the past 160 years, depiction of the full diversity of this phylum is lacking. To systematically describe the characteristics of these parasites, we created a database of 1,440 species and their attributes, including the hosts they infect and spore characteristics. We find that microsporidia have been reported to infect 16 metazoan and 4 protozoan phyla, with smaller phyla being underrepresented. Most species are reported to infect only a single host, but those that are generalists are also more likely to infect a broader set of host tissues. Strikingly, polar tubes are threefold longer in species that infect tissues besides the intestine, suggesting that polar tube length is a determinant of tissue specificity. Phylogenetic analysis revealed four clades which each contain microsporidia that infect hosts from all major habitats. Although related species are more likely to infect similar hosts, we observe examples of changes in host specificity and convergent evolution. Taken together, our results show that microsporidia display vast diversity in their morphology and the hosts they infect, illustrating the flexibility of these parasites to evolve new traits. IMPORTANCE Microsporidia are a large group of parasites that cause death and disease in humans and many agriculturally important animal species. To fully understand the diverse properties of these parasites, we curated species reports from the last 160 years. Using these data, we describe when and where microsporidia were identified and what types of animals and host tissues these parasites infect. Microsporidia infect hosts using a conserved apparatus known as the polar tube. We observe that the length of this tube is correlated with the tissues that are being infected, suggesting that the polar tube controls where within the animals that the parasite infects. Finally, we show that microsporidia species often exist in multiple environments and are flexible in their ability to evolve new traits. Our study provides insight into the ecology and evolution of microsporidia and provides a useful resource to further understand these fascinating parasites.

The present study reports a case of hepatic microsporidiosis caused by Microgemma sp in brackishwater fish, Boleophthalmus dussumieri (Valenciennes, 1837) (n = 60), from north-west coast of India. An eight-month study from September 2017 to April 2018 revealed a prevalence of 11.6% for this parasite. The microsporidian showed tissue-specific infection and did not reveal any gross pathology in infected fish. Large whitish cysts containing microspores of size 0.3–0.5 mm were observed in the liver of fish. The range of pyriform microsporidian spore size varied from 2.9–3.77 X 1.85–2.67 µm. Histological observations of infected liver revealed large xenoma of the microsporidian filled with spores and encircled by a cyst wall-like layer. Scanning electron microscopy of the spores showed a distinct groove on the anterior end of the spore for polar tube extrusion. Polymerase chain reaction (PCR) amplification of the DNA extracted from the microsporidian spores using primers targeting small ribosomal subunit DNA (SSU rDNA) yielded ~ 1340 bp amplicon and the genetic distance analysis showed a 0.2% variation with the reported M. tilanpasiri. Accordingly, in the phylogenetic tree, the present species of Microgemma clustered with M. tilanpasiri. Even though, the morphomeristic characters of the present Microgemma sp. was marginally different from the reported M. tilanpsasiri; the SSU rDNA showed considerably higher similarity with M. tilanpasiri. Thus, we report the species of Microgemma as Microgemma aff. tilanpasiri from a new host. This is the first report of a microsporidian from B. dussumieri and the first record of the genus Microgemma from India.

Background: The lizardfish is an economically important fish in the Persian Gulf with high rates of parasitic infections. Microsporidia species, as opportunistic parasites, cause several disorders, which in turn result in economic problems. Objectives: The main objective was to evaluate Heterosporis sp. infection in Persian Gulf lizardfish using the small subunit ribosomal RNA phylogenetics to describe and classification of the unknown microsporidia species as well as morphological characteristics. Methods: The abdominal cavities of fifty specimens of lizardfish, Saurida undosquamis, were examined using morphological and molecular techniques. Some irregular whitish cyst-like were fixed for histopathological and transmission electron observations. The small subunit ribosomal genomic DNA was studied and a 1,279 bp genomic sequence was amplified and investigated for molecular analysis. Results: Twenty-two (out of fifty) specimens were infected with irregular whitish microsporidian cysts. Light and electron microscopic findings revealed round cysts containing large numbers of monomorphic and ovoid spores with a posterior vacuole. Polar tube coiled between six and eight-times, in one row. The large xenoma (hypertrophied parasitizing host cells) was encapsulated by a host-derived thick connective tissue in pathological samples. The phylogenetic analysis showed that despite some morphological similarity of the Persian Gulf microsporidia sp. to Glugea spp., the most closely related species with minimum genetic distance to Heterosporis anguillarum isolated is Japanese eels (Anguillajaponica). Conclusions: This is the first phylogenetic report of microsporidian infections in mesenteric tissues of lizardfish S. undosquamis in Iran including morphological and molecular markers, to introduce novel species

Pollen grain is a unique haploid organism characterized by two key physiological processes: activation of metabolism upon exiting dormancy and polar tube growth. In gymnosperms and flowering plants, these processes occur in different time frames and exhibit important features; identification of similarities and differences is still in the active phase. In angiosperms, the growth of male gametophyte is directed and controlled by its microenvironment, while in gymnosperms it is relatively autonomous. Recent reviews have detailed aspects of interaction between angiosperm female tissues and pollen such as interactions between peptides and their receptors; however, accumulated evidence suggests low-molecular communication, in particular, through ion exchange and ROS production, equally important for polar growth as well as for pollen germination. Recently, it became clear that ROS and ionic currents form a single regulatory module, since ROS production and the activity of ion transport systems are closely interrelated and form a feedback loop.

Enterocytozoon hepatopenaei (EHP) is an obligate, intracellular, spore-forming parasite, which mainly infects the gastrointestinal tract of shrimp. It significantly hinders the growth of shrimp, which causes substantial economic losses in farming. In this study, we established and optimized a SYBR Green I fluorescent quantitative PCR (qPCR) assay based on the polar tube protein 2 (PTP2) gene for the quantitative analysis of EHP-infected shrimp. The result showed that the optimum annealing temperature was 60 &deg, C for the corresponding relation between the amplification quantitative (Cq) and the logarithmic of the initial template quantity (x), conformed to Cq = &minus, 3.2751x + 31.269 with a correlation coefficient R2 = 0.993. The amplification efficiency was 102%. This qPCR method also showed high sensitivity, specificity, and repeatability. Moreover, a microscopy method was developed to observe and count EHP spores in hepatopancreas tissue of EHP-infected shrimp using Fluorescent Brightener 28 staining. By comparing the PTP2-qPCR and microscopy method, the microscopic examination was easier to operate whereas PTP2-qPCR was more sensitive for analysis. And we found that there was a correspondence between the results of these two methods. In summary, the PTP2-qPCR method integrated microscopy could serve for EHP detection during the whole period of shrimp farming and satisfy different requirements for detecting EHP in shrimp farming.

Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 μm⋅s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection., Author summary Microsporidia infect a wide range of hosts: from economically important invertebrates such as silkworms and honey bees, to vertebrates including humans, where infection in immunocompromised patients can be fatal. In order to infect the host, microsporidia utilize a unique harpoon-like invasion organelle called the polar tube (PT), which serves as a pathway for transport of infectious cargo to the host. Knowledge of how the long PT is packaged in the spore, the kinetics of its firing and how the cargo is transported through the PT are still poorly understood. We use electron microscopy techniques to generate 3D reconstructions of intact spores, which reveal the configuration of the PT and other organelles within. Using high-speed live-cell imaging, we capture and quantify the kinetics of PT firing, and how cargo is deformed during transport through the narrow PT. Our study provides new insights into architecture and dynamics of the PT, which serve as foundations for our understanding of microsporidia infection.

Microsporidia are a large group of fungal-related obligate intracellular parasites. They are responsible for infections in humans as well as in agriculturally and environmentally important animals. Although microsporidia are abundant in nature, many of the molecular mechanisms employed during infection have remained enigmatic. In this review, we highlight recent work showing how microsporidia invade, proliferate and exit from host cells. During invasion, microsporidia use spore wall and polar tube proteins to interact with host receptors and adhere to the host cell surface. In turn, the host has multiple defence mechanisms to prevent and eliminate these infections. Microsporidia encode numerous transporters and steal host nutrients to facilitate proliferation within host cells. They also encode many secreted proteins which may modulate host metabolism and inhibit host cell defence mechanisms. Spores exit the host in a non-lytic manner that is dependent on host actin and endocytic recycling proteins. Together, this work provides a fuller picture of the mechanisms that these fascinating organisms use to infect their hosts.

Objective In this study, a north polar tube (Portex® North Polar tube [Ivory PVC Portex tube; Smiths Medical International, Hythe, United Kingdom]) (NPT) and spiral tube (ST) were compared for their ability to provide a nasal airway in patients during maxillofacial surgery. Methods Patients who were aged 18 to 65 years with American Society of Anaesthesiologists score 1 to 2 and Mallampati score 1 to 2 were included in the study. The anesthesia technique was standardized in all patients. Patients were divided into 2 groups randomly: the NPT (n = 35) and ST groups (n = 35). Anesthesia was administered with 1% to 2% sevoflurane and a mixture of 50% oxygen + 50% air in both groups. The mean arterial pressure and heart rate values of preinduction; preintubation; and the first, second, third, and fifth minutes of intubation were recorded. Epistaxis, intraoral bleeding, cuff burst, use of Magill pens, duration of intubation, intubation difficulty, laryngeal compression, head position change, and glottic grade were evaluated. Results There were no significant differences between groups in terms of demographic data (gender, age, height, weight, body mass index, American Society of Anaesthesiologists score, and Mallampati score). Macintosh laryngoscopes were used during intubation in all patients. There was no significant difference between groups in terms of intubation difficulty, duration of intubation, use of Magill pens, and determination of the glottic grade (P > 0.05). Epistaxis was significantly lower in the NPT group than in the ST group (P = 0.012). Intraoral bleeding was significantly higher in the ST group than in the NPT group (P = 0.001). During intubation, laryngeal compression (Sellick maneuver) and head position changes were significantly lower in the NPT group than in the ST group (P = 0.003 and P < 0.001, respectively). There were no significant differences in mean arterial pressure and heart rate between the 2 groups. Conclusions We conclude that the NPT was associated with less epistaxis and manipulations such as laryngeal compression and head position changes when compared with the ST during nasotracheal intubation. The velvet-soft polyvinyl chloride material of the NPT appears to be responsible for this advantage., Graphical abstract Image, graphical abstract

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

Encephalitozoon cuniculi is an important microsporidian pathogen that is considered an emergent, zoonotic, and opportunistic. It infects both domestic and laboratory rabbits, generating severe chronic interstitial and granulomatous nephritis with fibrosis and granulomatous encephalitis. Encephalitozoonosis is diagnosed in paraffin-embedded sections by examining the spores in the host tissues. The spores are difficult to observe when the samples are stained with hematoxylin and eosin (H&E), particularly when there is an inflammatory reaction and tissue damage. The spores are easily mistaken for other microorganisms, such as fungi (yeasts), protozoa, and bacteria. In our study, we used kidney samples from E. cuniculi–positive rabbits and employed 14 recommended histologic stains for detecting microsporidia spores: alcian blue, calcofluor white, Giemsa, Gram, Grocott, H&E, Luna, Luxol fast blue, Masson trichrome, modified trichrome stain (MTS), periodic acid–Schiff reaction (PAS), Van Gieson, Warthin–Starry (WS), and Ziehl–Neelsen (ZN).We concluded that MTS and Gram stain, detected by light microscopy, and calcofluor white stain, detected by ultraviolet light microscopy, are the best stains for detecting spores of E. cuniculi in paraffin-embedded tissues from infected rabbits. These stains were superior to WS, ZN, Giemsa, and PAS for identifying spores without background “noise” or monochromatic interference. Also, they allow individual spores to be discerned in paraffin-embedded tissues. MTS allows observation of the polar tube, polaroplast, and posterior vacuole, the most distinctive parts of the spore.

Microsporidia are important opportunistic human pathogens in immune-suppressed individuals, such as those with HIV/AIDS and recipients of organ transplants. The sporoplasm is critical for establishing microsporidian infection. Despite the biological importance of this structure for transmission, there is limited information about its structure and composition that could be targeted for therapeutic intervention. Here, we identified a novel E. hellem sporoplasm surface protein, EhSSP1, and demonstrated that it can bind to host cell mitochondria via host VDAC. Our data strongly suggest that the interaction between SSP1 and VDAC is important for the association of mitochondria with the parasitophorous vacuole during microsporidian infection. In addition, binding of SSP1 to the host cell is associated with the final steps of invasion in the invasion synapse., Microsporidia are opportunistic intracellular pathogens that can infect a wide variety of hosts ranging from invertebrates to vertebrates. During invasion, the microsporidian polar tube pushes into the host cell, creating a protective microenvironment, the invasion synapse, into which the sporoplasm extrudes. Within the synapse, the sporoplasm then invades the host cell, forming a parasitophorous vacuole (PV). Using a proteomic approach, we identified Encephalitozoon hellem sporoplasm surface protein 1 (EhSSP1), which localized to the surface of extruded sporoplasms. EhSSP1 was also found to interact with polar tube protein 4 (PTP4). Recombinant EhSSP1 (rEhSSP1) bound to human foreskin fibroblasts, and both anti-EhSSP1 and rEhSSP1 caused decreased levels of host cell invasion, suggesting that interaction of SSP1 with the host cell was involved in invasion. Coimmunoprecipitation (Co-IP) followed by proteomic analysis identified host cell voltage-dependent anion channels (VDACs) as EhSSP1 interacting proteins. Yeast two-hybrid assays demonstrated that EhSSP1 was able to interact with VDAC1, VDAC2, and VDAC3. rEhSSP1 colocalized with the host mitochondria which were associated with microsporidian PVs in infected cells. Transmission electron microscopy revealed that the outer mitochondrial membrane interacted with meronts and the PV membrane, mitochondria clustered around meronts, and the VDACs were concentrated at the interface of mitochondria and parasite. Knockdown of VDAC1, VDAC2, and VDAC3 in host cells resulted in significant decreases in the number and size of the PVs and a decrease in mitochondrial PV association. The interaction of EhSSP1 with VDAC probably plays an important part in energy acquisition by microsporidia via its role in the association of mitochondria with the PV.

Microsporidia are eukaryotic unicellular parasites that have been studied for more than 150 years. They are found throughout the world and are capable of infecting various invertebrate and vertebrate hosts. They can cause disease in both immune-compromised and immune-competent humans. In immune-compromised individuals, infections can be severe and often fatal. Microsporidia possess a unique, highly specialized invasion mechanism that involves a structure known as the polar tube as well as the spore wall. During spore germination, the polar tube rapidly discharges from the spore and deliver the sporoplasm into the host cell. Spores are the only stage of microsporidia that can survive outside of host cells. Since the first attempt to culture microsporidia in vitro in 1930s, their cultivation has served a critical role in the study and diagnosis of these parasites. In this chapter, we include methods on the cultivation, isolation, and cryopreservation of Encephalitozoon cuniculi, which can infect humans and provides a useful model for other microsporidia. These methods can also be utilized for the culture of Encephalitozoon hellem or Encephalitozoon intestinalis. © 2018 by John Wiley & Sons, Inc.

The 14th International Workshops on Opportunistic Protists (IWOP-14) was held August 10-12, 2017 in Cincinnati, OH, USA. The IWOP meetings focus on opportunistic protists (OIs); for example, free-living amoebae, Pneumocystis spp., Cryptosporidium spp., Toxoplasma, the Microsporidia, and kinetoplastid flagellates. The highlights of Pneumocystis spp. research included the reports of primary homothallism for mating; a potential requirement for sexual replication in its life cycle; a new antigen on the surface of small asci; roles for CLRs, Dectin-1, and Mincle in host responses; and identification of MSG families and mechanisms used for surface variation. Studies of Cryptosporidia spp. included comparative genomics, a new cryopreservation method; the role of mucin in attachment and invasion, and epidemiological surveys illustrating species diversity in animals. One of the five identified proteins in the polar tube of Microsporidia, PTP4, was shown to play a role in host infection. Zebrafish were used as a low cost vertebrate animal model for an evaluation of potential anti-toxoplasma drugs. Folk medicine compounds with anti-toxoplasma activity were presented, and reports on the chronic toxoplasma infection provided evidence for increased tractability for the study of this difficult life cycle stage. Escape from the parasitophorus vacuole and cell cycle regulation were the topics of the study in the acute phase.

© 2015 Elsevier Inc.Microsporidia are highly divergent fungi that are obligate intracellular pathogens of a wide range of host organisms. Here we review recent findings from the genome sequences of mosquito-infecting microsporidian species Edhazardia aedis and Vavraia culicis, which show large differences in genome size, although similar numbers of predicted genes. We also show a video of E. aedis polar tube firing, which is the dramatic mechanism used by microsporidia to deliver the germ cell (sporoplasm) into the host cell to initiate intracellular infection.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene and is conserved in a wide range of eukaryotic organisms. The RNAi mechanism has provided unique opportunities in combating honey bee diseases caused by various parasites and pathogens. Nosema ceranae is a microsporidian parasite of European honey bees, Apis mellifera, and has been associated with honey bee colony losses in some regions of the world. Here we explored the possibility of silencing the expression of a N. ceranae putative virulence factor encoding polar tube protein 3 (ptp3) which is involved in host cell invasion as a therapeutic strategy for controlling Nosema parasites in honey bees. Our studies showed that the oral ingestion of a dsRNA corresponding to the sequences of N. ceranae ptp3 could effectively suppress the expression of the ptp3 gene in N. ceranae infected bees and reduce Nosema load. In addition to the knockdown of ptp3 gene expression, ingestion of ptp3-dsRNA also led to improved innate immunity in bees infected with N. ceranae along with an improvement in physiological performance and lifespan compared to untreated control bees. These results strongly suggest that RNAi-based therapeutics hold real promise for the effective treatment of honey bee diseases in the future and warrant further investigation.

Microsporidia are obligate intracellular, spore-forming, fungal-related pathogens that employ a unique organelle, the polar tube, to transfer infectious spore contents into host cells to initiate infection. Spore adherence to host cells may provide the proximity required for polar tube/host cell interaction during in vivo infection. In previous in vitro studies, host sulfated glycosaminoglycans (GAGs) or recombinant microsporidia endospore protein (EnP1) was implicated in the pathogen adherence and infection process; however, complete ablation of spore adherence and infection could not be achieved, suggesting that additional or alternative spore and host cell determinants of adherence and infection may exist. Analysis of the Encephalitozoon intestinalis genome revealed about 100 predicted proteins containing the canonical integrin-binding motif arginine-glycine-aspartic acid (RGD); and, many pathogens have been shown to engage integrin molecules on cell surfaces. We hypothesized that host cell integrins play a role in microsporidia adherence and infection. In this study, we demonstrated that addition of exogenous integrin ligands or recombinant alpha 3 beta 1 integrin or alpha 5 beta 1 integrin to assays of E. intestinalis adherence and infection significantly reduced spore adherence and infection of host cells, supporting our hypothesis and implicating these specific integrins as putative host cell receptors for E. intestinalis spores.

All microsporidia possess a unique, highly specialized invasion mechanism that involves the polar tube and spore wall. The interaction between spore wall proteins (SWPs) and polar tube proteins (PTPs) in the formation, arrangement, orderly orientation, and function of the polar tube and spore wall remains to be determined. This study was undertaken to examine the protein interactions of Nosema bombycis SWP7 (NbSWP7), NbSWP9, and PTPs. Coimmunoprecipitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and yeast two-hybrid data demonstrated that NbSWP9, but not NbSWP7, interacts with NbPTP1 and NbPTP2. Furthermore, immunoelectron microscopy (IEM) showed that NbSWP9 was localized mainly in the developing polar tube of sporoblasts, while NbSWP7 was found randomly in the cytoplasm. However, both NbSWP9 and NbSWP7 were located in the polar tube and spore wall of N. bombycis mature spores. The reason why NbSWP7 was localized to the polar tube may be due to the interaction between NbSWP9 and NbSWP7. Interestingly, the majority of NbSWP9, but not NbSWP7, accumulated in the beginning part of the extruded polar tube and the ruptured spore wall called the anchoring disk (AD) when the mature spores germinated under weak-alkaline environmental stimulation. Additionally, anti-NbSWP9 antibody reduced spore germination in a dose-dependent manner. In conclusion, our study further confirmed that NbSWP9 is a scaffolding protein that not only anchors and holds the polar tube but also tethers the polar tube to the spore wall.

In nature, the population of Nosema bombycis (Microsporidia) causing pebrine disease is small and their development is extremely slow and only few ultimately producing spores. Pebrine infected silkworm, Bombyx mori larvae collected from sericulture field were alive till 3rd generation though the concentration of N.bombycis spore was very high (2.4 - 3.0 x 108 spores. mL-1). All larvae were died during 4th generation with extremely high concentration of pebrine spores (3.0 - 4.0 x 109 spores. mL-1) and mostly contain long polar tube (LT). Alternately, all larvae were died immediately (at 3rd stage of 1st generation) when it was artificially inoculated with same concentration of N.bombycis spores harvested from field (2.4 - 3.0 x 108 spores. mL-1) though concentration of spores harvest was very less (3.0- 4.0 x 106 spores. mL-1) and mostly contain short polar tube (ST). Artificially pebrine infected male moth when mated with healthy female moth took six generations to develop pebrine disease and all larvae were died at the 2nd stage with very less spore harvest (3.0 - 10.0 x 105 spores. mL -1). Survival percentage was increased in all generations (~92.0% at 4th generation) when silkworm rearing was conducted under new integrated disease management system.

International audience; Microsporidia are obligate intracellular eukaryotic parasites with a broad host spectrum characterized by a unique and highly sophisticated invasion apparatus, the polar tube (PT). In a previous study, two PT proteins, named AlPTP1 (50 kDa) and AlPTP2 (35 kDa), were identified in Antonospora locustae, an orthoptera parasite that is used as a biological control agent against locusts. Antibodies raised against AlPTP2 cross-reacted with a band migrating at ~70 kDa, suggesting that this 70-kDa antigen is closely related to AlPTP2. A blastp search against the A. locustae genome database allowed the identification of two further PTP2-like proteins named AlPTP2b (568 aa) and AlPTP2c (599 aa). Both proteins are characterized by a specific serine- and glycine-rich N-terminal extension with elastomeric structural features and share a common C-terminal end conserved with AlPTP2 (~88% identity for the last 250 aa). MS analysis of the 70-kDa band revealed the presence of AlPTP2b. Specific anti-AlPTP2b antibodies labelled the extruded PTs of the A. locustae spores, confirming that this antigen is a PT component. Finally, we showed that several PTP2-like proteins are also present in other phylogenetically related insect microsporidia, including Anncaliia algerae and Paranosema grylli.

Encephalitozoon cuniculi is a microsporidian species which can induce subclinical to serious disease in mammals including rabbits, a definitive natural host. The pathophysiology of infection has not been comprehensively elucidated. In this exploratory study, we utilized two mass spectrometry approaches: first, the analysis of the humoral response by profiling the microsporidian antigens as revealed by Western blot screening, and second, implementing the iTRAQ ®-labeling protocol to focus on the changes within the host proteome during infection. Seven E. cuniculi proteins were identified at one-dimensional gel regions where specific seropositive reaction was observed by Western blot, including polar tube protein 3, polar tube protein 2, and for the first time reported: heat shock related 70kDa protein, poly-saccharide deacetylase domain-containing protein, zinc finger protein, spore wall and anchoring disk complex protein EnP1, and translation elongation factor 1 alpha. In addition, there was a significant increase of nine host proteins in blood samples from E. cuniculi-diseased rabbits in comparison with non-diseased control subjects undergoing various inflam-matory processes. This included serum paraoxonase, alpha-1-antiproteinase F precursor and alpha-1-antiproteinase S-1 which have presumptive catalytic activity likely related to infection control, and cystatin fetuin-B-type, an enzyme regulator that has been poorly studied to date. Notably, 11 proteins were found to be statistically increased in rabbits with neurological versus renal clinical presentation of E. cuniculi infection. Overall, this novel analysis based on mass spectrometry has provided new insights on the inflammatory and humoral responses during E. cuniculi infection in rabbits.

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

Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens., Author summary Microsporidia are obligate intracellular parasites that cause disease in immune suppressed individuals such as those with HIV/AIDS and recipients of organ transplants. The microsporidia are defined by a unique invasion organelle, the polar tube. The formation of this organelle and its role in the mechanism of infection remain unknown. Herein, we have identified a role for Encephalitozoon hellem polar tube protein 4 (PTP4) in infection demonstrating that PTP4 can bind to the host cell surface via the host transferrin receptor 1 (TfR1) protein. Interfering with the interaction of PTP4 and TfR1 causes a significant decrease in microsporidian infection of host cells. These data suggest that PTP4 functions as an important microsporidian protein during host cell infection by this pathogen.

Microsporidia are intracellular organisms of increasing importance as opportunistic pathogens in immunocompromised patients. Host cells are infected by the extrusion and injection of polar tubes located within spores. The spore is surrounded by a rigid spore wall which, in addition to providing mechanical resistance, might be involved in host cell recognition and initiation of the infection process. A 51-kDa outer spore wall protein was identified in Encephalitozoon cuniculi with the aid of a monoclonal antibody, and the corresponding gene, SWP1 , was cloned by immunoscreening of a cDNA expression library. The cDNA encodes a protein of 450 amino acids which displays no significant similarities to known proteins in databases. The carboxy-terminal region consists of five tandemly arranged glycine- and serine-rich repetitive elements. SWP1 is a single-copy gene that is also present in the genomes of Encephalitozoon intestinalis and Encephalitozoon hellem as demonstrated by Southern analysis. Indirect immunofluorescence and immunoelectron microscopy revealed that SWP1 is differentially expressed during the infection cycle. The protein is absent in replicative meronts until 24 h postinfection, and its expression is first induced in early sporonts at a time when organisms translocate from the periphery to the center of the parasitophorous vacuole. Expression of SWP1 appears to be regulated at the mRNA level, as was shown by reverse transcriptase PCR analysis. Further identification and characterization of stage-specific genes might help to unravel the complex intracellular differentiation process of microsporidia.

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

A multiplex bead assay (MBA) was used to analyze serum samples collected longitudinally from children enrolled in a drug trial for treatment of filariasis in Leogane, Haiti. Recombinant antigens Bm14 and Bm33 from Brugia malayi, third polar tube protein (PTP3) from Encephalitozoon cuniculi, and merozoite surface protein-1(19) (MSP-1(19)) from Plasmodium falciparum were coupled to carboxylated polystyrene microspheres. IgG responses to PTP3 and MSP-1(19) were not affected by albendazole (ALB), diethylcarbamazine (DEC), or combination of diethylcarbamazine and albendazole (DEC/ALB). However, IgG and IgG4 responses to Bm14 and Bm33 were significantly decreased (P < 0.001) by DEC and DEC/ALB treatment. Antibody responses to Bm14 and Bm33 decreased after DEC treatment (but not placebo) among children who were negative for microfilaremia and antigenemia at baseline, suggesting that these children harbored early stages of infection. The MBA is an excellent serologic technique for multiple antigens that offers substantial advantages over single-antigen based enzyme-linked immunosorbent assay in mass drug administration studies for monitoring changes in antibody levels.

To infect their host cells the Microsporidia use a unique invasion organelle, the polar tube complex. During infection, the organism is injected into the host cell through the hollow polar tube formed during spore germination. Currently, three proteins, PTP1, PTP2, and PTP3 have been identified by immunological and molecular techniques as being components of this structure. Genomic data suggests that Microsporidia are capable of O-linked, but not N-linked glycosylation as a post-translational protein modification. Cells were infected with Encephalitozoon cunicuili, labeled with radioactive mannose or glucosamine, and the polar tube proteins were examined for glycosylation. PTP1 was clearly demonstrated to be mannosylated consistent with 0-glycosylation. In addition, it was evident that several other proteins were mannosylated, but no labeling was seen with glucosamine. The observed post-translational mannosylation of PTP1 may be involved in the functional properties of the polar tube, including its adherence to host cells during penetration.

The Kingdom Fungi, home to molds, mushrooms, lichens, rusts, smuts and yeasts, comprises eukaryotes with remarkably diverse life histories that make essential contributions to the biosphere, human industry, medicine and research. With the aim of enticing biologists to include fungi in their research, we note that many fungi have haploid genetics, and that those in cultivation are essentially immortal, two features that make it easier to associate traits with genotype, even for complex traits, than with Drosophila or Arabidopsis. The typical fungal genome size of 30–40 Mb is small by eukaryotic standards, which is why fungi have led the way as models for eukaryote genome sequencing with over 100 assembled genome sequences available [1, 2]. For some fungi, DNA transformations, gene knockouts and knockdowns are routine. Species of Ascomycota and Basidiomycota show simple, multicellular development with differentiated tissues. In many species these tissues are large enough to support studies of transcription and translation in the lab and even in nature. About a billion years ago, give or take 500 million years [3], a population of aquatic, unicellular eukaryotes making sporangia containing zoospores each with a single posterior flagellum split into two lineages: one eventually gave rise to animals, the other to fungi. Here we shall summarize the major diversifications of the Fungi by introducing each major fungal branch in the order that it is thought to have diverged (Figure 1) and presenting salient facts about fungal modes of nutrition, reproduction, communication and interaction with other life. Our views are strongly influenced by the Fungal Tree of Life Project [4–6]. Readers interested in learning more about fungi are encouraged to consult any of a number of comprehensive texts [7, 8]. Figure 1 The fungi. Phylogenetic tree, based on [4], showing relationships of many of the fungal lineages fit to geologic time using the program r8s [73] and considering Paleopyrenomycites to be a member of the Ascomycota [3]. Arrows depict changes in morphology ... The exact order of divergence in deep regions of the eukaryotic tree is controversial. On the lineage that leads to the Fungi there are thought to be two other groups; the first to diverge are the nucleariid amoebae [9], and the next the Microsporidia. Microsporidia are either the sister group to the Fungi, or lie within the Fungi, (Figure 1), and they should be included in studies of fungi. They are unculturable, obligate parasites of animals, including humans. They have extremely reduced eukaryotic genomes — with a genome size of ~2.6 Mb and ~2000 genes [10, 11] — remnant mitochondria, and unique morphologies related to parasitism, including a very frightening polar tube used to initiate infection [10].

A captive, adult male Goeldi's monkey (Callimico goeldii) (GM) presented in acute respiratory distress 4 yr after importation into the United States from Europe. Radiographs and echocardiogram were consistent with heart failure. The monkey died within 24 hr of presentation. Necropsy findings included multicentric arteritis and aortitis with aneurysm associated with microsporidian organisms morphologically consistent with Encephalitozoon species. Polymerase chain reaction confirmed organisms were Encephalitozoon cuniculi. Sequence analysis of amplicons generated by using primers specific for the polar tube protein of E. cuniculi determined the organism to be genotype II. An E. cuniculi serosurvey of potentially exposed conspecifics that represented approximately 40% of the captive GM population in the United States was conducted. Multiple individuals that had been imported from Europe with the individual of this report were seropositive via an immunofluorescent antibody assay for E. cuniculi. Multiple samples were available from 3 individuals that demonstrated a decrease in titer or reversion to seronegative status within 3 yr of initial positive status. All other GM were negative on serology. This case is unique in that the genotype identified (genotype II) was different than the genotype (genotype III) reported in other New World primate (NWP) species, the patterns of arteritis were different from the typical pattern of microsporidial vasculitis described in other species, and clinical disease was observed in an adult. Most reported cases of clinical disease secondary to E. cuniculi in NWP have been in neonates and juveniles.

The microsporidian Encephalitozoon cuniculi is a spore-forming, obligate, intracellular parasitic pathogen with a unique organelle called a polar tube, the extrusion of which is essential for invading a host cell. The polar tube consists of three proteins: polar tube protein 1 (PTP1), PTP2, and PTP3. We established three mouse monoclonal antibodies (MAb1, MAb2, and MAb4) against E. cuniculi PTP1. An enzyme-linked immunosorbent assay (ELISA) indicated that all three MAbs reacted with the outer surface of extruded polar tubes and that they also strongly bound to an intracellular antigen in infected host cells. Two-dimensional (2-D) immunoblot analysis showed that MAb1 and MAb2 recognized PTP1 spots at 52 kDa and some spotty smears at molecular weights of less than 50 kDa, whereas MAb4 recognized only PTP1 spots at 52 kDa. Interestingly, all three MAbs were of the immunoglobulin (Ig) E class, suggesting that, in addition to the highly immunogenic or antigenic nature, the PTP1 antigen may have the potential to induce specific IgE antibody production in mice. These antibodies may be useful in the study of allergenic PTP1 as well in the purification and detection of the PTP1 antigen.

A generalized mathematical treatment is postulated to investigate the condensation heat transfer performance of horizontal tubes with varying geometries, with the presence of non-condensable gases in the free stream. The governing equations of mass, momentum and energy conservation in the liquid phase are solved semi-analytically, with matching constraints being imposed at the liquid–vapour interface, while the governing equations of energy and species conservation in the vapour phase are solved numerically. An air–water vapour system is considered for demonstrating the mathematical model. Special cases of the model are illustrated with the aid of elliptical and equiangular spiral geometries. It is revealed that the geometrical features of typical polar surfaces can turn out to be favourable in arresting probable drastic reductions in the condensation heat transfer rates that could be otherwise associated with the presence of non-condensable gases in the free stream. The favourable effects induced by polar surfaces become relatively more prominent, as percentage of non-condensable gases in the free stream increases. A geometrical shape function is also ascertained in this regard, which quantifies the extent of this augmentation in the heat transfer performance. In general, it is suggested that polar surfaces with higher values of the shape function over a majority of the azimuthal regime can turn out to be more desired choices for achieving enhanced rates of condensation heat transfer, provided that there are no serious manufacturability constraints.

The flow of a matter, accreting onto a magnetized neutron star, is accompanied by an electric current. The closing of the electric current occurs in the crust of a neutron stars in the polar region across the magnetic field. But the conductivity of the crust along the magnetic field greatly exceeds the conductivity across the field, so the current penetrates deep into the crust down up to the super conducting core. The magnetic field, generated by the accretion current, increases greatly with the depth of penetration due to the Hall conductivity of the crust is also much larger than the transverse conductivity. As a result, the current begins to flow mainly in the toroidal direction, creating a strong longitudinal magnetic field, far exceeding an initial dipole field. This field exists only in the narrow polar tube of $r$ width, narrowing with the depth, i.e. with increasing of the crust density $\rho$, $r\propto \rho^{-1/4}$. Accordingly, the magnetic field $B$ in the tube increases with the depth, $B\propto \rho^{1/2}$, and reaches the value of about $10^{17}$ Gauss in the core. It destroys super conducting vortices in the core of a star in the narrow region of the size of the order of ten centimeters. Because of generated density gradient of vortices they constantly flow into this dead zone and the number of vortices decreases, the magnetic field of a star decreases as well. The attenuation of the magnetic field is exponential, $B=B_0(1+t/\tau)^{-1}$. The characteristic time of decreasing of the magnetic field $\tau$ is equal to $\tau\simeq 10^3$ years. Thus, the magnetic field of accreted neutron stars decreases to values of $10^8 - 10^9$ Gauss during $10^7-10^6$ years., Comment: 8 pages,6 figures

Microsporidia form environmentally resistant spores that are critical for their host-to-host transmission and persistence in the environment. The spore walls of these organisms are composed of two layers, the exospore and the endospore. Two spore wall proteins (SWP1 and SWP2) have been previously identified in members of the Encephalitozoonidae family. These proteins localize to the exospore. The endospore is known to contain chitin, and a putative glycosylphosphatidylinositol (GPI)-anchored chitin deacetylase has been localized to the plasmalemma-endospore interface. Using proteomic techniques, we have identified a new spore wall protein (SWP3) that is located in the endospore. The gene for this protein is located on chromosome 1 and corresponds to the open reading frame ECU01_1270. SWP3 is predicted to have a signal peptide and to be GPI anchored. Consistent with these modifications, two-dimensional electrophoresis demonstrated that SWP3 has an acidic pI and a molecular mass of

Brachiola algerae (Vavra et Undeen, 1970) Lowman, Takvorian et Cali, 2000, originally isolated from a mosquito, has been maintained in rabbit kidney cells at 29 degrees C in our laboratory. This culture system has made it possible to study detailed aspects of its development, including spore activation, polar tube extrusion, and the transfer of the infective sporoplasm. Employing techniques to ultrastructurally process and observe parasite activity in situ without disturbance of the cultures has provided details of the early developmental activities of B. algerae during timed intervals ranging from 5 min to 48 h. Activated and nonactivated spores could be differentiated by morphological changes including the position and arrangement of the polar filament and its internal structure. The majority of spores extruded polar tubes and associated sporoplasms within 5 min post inoculation (p.i.). The multilayered interlaced network (MIN) was present in extracellular sporoplasms and appeared morphologically similar to those observed in germination buffer. Sporoplasms, observed inside host cells were ovoid, contained diplokaryotic nuclei, vesicles reminiscent of the MIN remnants, and their plasmalemma was already electron-dense with the "blister-like" structures, typical of B. algerae. By 15 min p.i., the first indication of parasite cell commitment to division was the presence of chromatin condensation within the diplokaryotic nuclei, cytoplasmic vesicular remnants of the MIN were still present in some parasites, and early signs of appendage formation were present. At 30 min p.i., cell division was observed, appendages became more apparent, and some MIN remnants were still present. By two hours p.i., the appendages became more elaborate and branching, and often connected parasite cells to each other. In addition to multiplication of the organisms, changes in parasite morphology from small oval cells to larger elongated "more typical" parasite cells were observed from 5 h through 36 h p.i. Multiplication of proliferative organisms continued and sporogony was well underway by 48 h p.i., producing sporonts and sporoblasts, but not spores. The observation of early or new infections in cell cultures 12-48 h p.i., suggests that there may also exist a population of spores that do not immediately discharge, but remain viable for some period of time. In addition, phagocytized spores were observed with extruded polar tubes in both the host cytoplasm and the extracellular space, suggesting another means of sporoplasm survival. Finally, extracellular discharged sporoplasms tightly abutted to the host plasmalemma, appeared to be in the process of being incorporated into the host cytoplasm by phagocytosis and/or endocytosis. These observations support the possibility of additional methods of microsporidian entry into host cells and will be discussed.