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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.

Nosema ceranae infection in Apis mellifera acts as a virulent pathogenic factor of honeybee colony collapse. Fumagillin is the only effective antimicrobial agent currently used against the microsporidial parasite N. ceranae. However, the toxicity of fumagillin is a concern. Therefore, in the present study, the screening of potent alternatives for fumagillin was performed for a total of 21 compounds, including 15 phenolics and 6 monoterpenes, based on their structure–activity correlation. The anti-nosemosis activity of each compound was determined by the relative inhibition level against the expression of the N. ceranae virulence factor encoding the polar tube protein 3 (ptp3) mRNA of the ptp3 gene. Each compound was preliminarily evaluated at 100 and 200 µM treatment concentrations. Then, the activity of 12 compounds showing activity at 200 µM was additionally determined at 40 µM, a concentration generally used in apiaries. At this concentration, gallol (benzene-1,2,3-triol) and 3-pentylcatechol (PC) showed higher activities than fumagillin, and 4-pentylgallol (PG) exhibited activity at a similar level to fumagillin. All three compounds selected in this study were low-molecular-weight phenolics. Among them, PC and PG, chemically synthesized compounds, commonly possess a hydrocarbon side chain of the pentyl group (-C5H11) in catechol and gallol, respectively. That is, the three treatment compounds were selected as potential beneficial alternatives for fumagillin and were markedly different from fumagillin in terms of their chemical structure. These results may trigger further extensive research on the biological mechanism for the prevention of nosemosis.

The species Metchnikovella dogieli (Paskerova et al. Protistology 10:148-157, 2016) belongs to one of the early diverging microsporidian groups, the metchnikovellids (Microsporidia: Metchnikovellidae). In relation to typical ('core') microsporidia, this group is considered primitive. The spores of metchnikovellids have no classical polar sac-anchoring disk complex, no coiled polar tube, no posterior vacuole, and no polaroplast. Instead, they possess a short thick manubrium that expands into a manubrial cistern. These organisms are hyperparasites; they infect gregarines that parasitise marine invertebrates. M. dogieli is a parasite of the archigregarine Selenidium pygospionis (Paskerova et al. Protist 169:826-852, 2018), which parasitises the polychaete Pygospio elegans. This species was discovered in samples collected in the silt littoral zone at the coast of the White Sea, North-West Russia, and was described based on light microscopy. No molecular data are available for this species, and the publicly accessible genomic data for metchnikovellids are limited to two species: M. incurvata Caullery & Mesnil, 1914 and Amphiamblys sp. WSBS2006. In the present study, we applied single-cell genomics methods with whole-genome amplification to perform next-generation sequencing of M. dogieli genomic DNA. We performed a phylogenetic analysis based on the SSU rRNA gene and reconstructed a multigene phylogeny using a concatenated alignment that included 46 conserved single-copy protein domains. The analyses recovered a fully supported clade of metchnikovellids as a basal group to the core microsporidia. Two members of the genus Metchnikovella did not form a clade in our tree. This may indicate that this genus is paraphyletic and requires revision.

Bombyx mori is an insect of economic importance in the production of silk. It often gets infected by Nosema bombycis, an intracellular parasite. The infection causes a fatal disease known as a Pebrine which affects the development of the worm. The infected larvae of silkworms are coated with brown spots and are unable to spin the silkworm thread. They lose appetite, become sluggish, opaque and ultimately die. The Spore Wall Protein 5 is an exospore protein in N. bombycis and interacts with the polar tube proteins PTP2 and PTP3, a part of the extrusion apparatus that facilitates infection of the host. SWP5 also plays an essential part in maintaining the structural integrity of the spore wall and could possibly regulate the route of the infection in N. bombycis. In the present study, the homology modelling of three protein structures SWP5, PTP2 and PTP3 were performed. The protein-protein interaction was studied and a complete complex of SWP5, PTP2 and PTP3 was generated to understand the discharge of the penetrating polar tube. Virtual screening and molecular dynamics simulation was performed and a potential lead-like molecule is identified.Communicated by Ramaswamy H. Sarma.

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae-infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.

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.

International audience; microsporidia are obligate intracellular pathogens capable of infecting a wide variety of hosts ranging from invertebrates to vertebrates. the infection process requires a step of prior adherence of microsporidia to the surface of host cells. a few studies demonstrated the involvement of proteins containing a ricin-b lectin (rbl) domain in parasite infection. in this study anncalia algerae and encephalitozoon cuniculi genomes were screened by bioinformatic analysis to identify proteins with an extracellular prediction and possessing rbl-type carbohydrate-binding domains, being both potentially relevant factors contributing to host cell adherence. three proteins named aarbll-1 and aarbll-2 from a. algerae and ecrbll-1 from e. cuniculi, were selected and comparative analysis of sequences suggested their belonging to a multigenic family, with a conserved structural rbl domain despite a significant amino acid sequence divergence. the production of recombinant proteins and antibodies against the three proteins allowed their subcellular localization on the spore wall and/or the polar tube. adherence inhibition assays based on pre-treatments with recombinant proteins or antibodies highlighted the significant decrease of the proliferation of both e. cuniculi and a. algerae, strongly suggesting that these proteins are involved in the infection process.

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)

Nosema bombycis (Nb) is a deadly species of microsporidia capable of causing pébrine, leading to heavy losses in sericulture. Germination is an important biological event in the invasion process of microsporidia. Septins, a family of membrane-associated proteins, play a critical role in tissue invasion and have been recognized as a virulence factor in numerous pathogens. Previous work in our laboratory has shown that Nosema bombycis septin2 (Nbseptin2) interacts with subtilisin-like protease 2 (NbSLP2). Herein, we found that Nbseptin2 was mainly associated with the plasma membrane in spores. Following spore germination, Nbseptin2 was found to co-localize with polar tube protein 1 (NbPTP1) at the polar cap and proximal zone of the polar tube. Co-immunoprecipitation and yeast two-hybrid analysis further confirmed that Nbseptin2 interacted with NbPTP1. The translocation and interaction of Nbseptin2 in the spores suggest that Nbseptin2 may play a significant role in microsporidia polar tube extrusion process. Our findings improve understanding of the mechanisms underlying microsporidia germination.

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.

Nosema ceranae is the most prevalent endoparasite of Apis mellifera iberiensis and it is a major health problem for bees worldwide. The infective capacity of N. ceranae has been demonstrated experimentally in honey bee brood, however no data are available about its prevalence in brood under natural conditions. Thus, brood combs from 10 different hives were analyzed over two consecutive years, taking samples before and after winter. A total of 1433 larvae/pupae were analyzed individually and N. ceranae (3.53%) was the microsporidian most frequently detected, as opposed to Nosema apis (0.42%) which was more frequently detected in conjunction with N. ceranae (0.71%). The active multiplication of both microsporidians was confirmed by the expression (real-time-PCR) of the N. ceranae polar tube protein 3 gene and/or the N. apis RNA polymerase II gene in 24% of the brood samples positive for Nosema spp. Both genes are related to microsporidian multiplication. As such, N. ceranae multiplication was confirmed in 1.06% of the samples, while N. apis multiplication was only observed in co-infections with N. ceranae (0.07%). Brood cells were analyzed for the presence of Nosema spp., as those are the immediate environment where the brood stages develop. The brood samples infected by Nosema spp. were in brood cells in which that microsporidians were not detected, while brood cells positive for N. ceranae hosted brood stages that were not apparently infected, indicating that this is unlikely to be the main pathway of infection. Finally, the colonies with brood infected by N. ceranae showed higher levels (numbers) of infected adult bees, although the differences were not significant before (P = 0.260), during (P = 0.055) or after (P = 0.056) brood sampling. These results show that N. ceranae is a bee parasite ubiquitous to all members of the colony, irrespective of the age of the bee. It is also of veterinary interest and should be considered when studying the epidemiology of the disease.

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.

Encephalitozoon cuniculi is a microsporidian which is frequently reported from rabbits. This microorganism can either ravage rabbit farms or transmit to humans from pet rabbits. This study aimed to investigate the prevalence and the genotype distribution of E. cuniculi among pet rabbits. In this study urine samples were collected from 50 pet rabbits, aged 2 months to 3 years, admitted to teaching veterinary hospital. Four races Lop, Dutch, Mix, and Angora were screened for E. cuniculi. The clinical symptoms were recorded and total DNA was extracted from urine samples. E. cuniculi was identified using amplification of the small subunit ribosomal RNA (ssu rRNA) gene and its genotypes were characterized using PCR/sequencing of the polar tube protein (PTP) gene. Phylogenetic tree was drawn to confirm the characterized genotypes. Out of 50 samples, 41 (82 %) of rabbits were asymptomatic, while nine (18 %) had at least one of symptoms including head-tilt, circling, and ataxia. A statistical correlation was seen between mean age + SD and symptoms (P-value = 0.039). The presence of E. cuniculi was confirmed in 16/50 (32 %) rabbits and all of them were identified as the genotype I. Our findings represented no consistency between E. cuniculi PCR – positive and the presence of symptoms (P-value = 0.318). Our results showed positive correlation between symptoms and age; however, the lack of correlation between PCR results with age may signify the latent infection in younger rabbits. All identified E. cuniculi were the genotype I, which is reported from rabbits and humans, highlighting the zoonotic concern for this genotype, particularly among subjects who keep pet rabbits.

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.

Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is a highly prevalent and hard to eradicate parasite in major shrimp aquaculture hubs. Laboratory models to study the spread of EHP have revealed horizontal transmission via spores released from infected shrimp into rearing water. However, infectivity duration for the released spores is unknown. Thus, we carried out experiments using aquaria in which shrimp heavily-infected with EHP were held for 4 weeks after which the presence of spores in the water was confirmed by SybrGreen qPCR, and spore activity was determined by polar tube extrusion assay. After the shrimp were removed, the EHP-water was used directly (100%), diluted 50% or diluted 75% before adding naive PL-12 and rearing for 16 days. Infection was determined by PCR and in situ hybridization (ISH) and compared to the results obtained using a standard EHP-cohabitation model. Infections developed at all dilutions by day 12 (100, 66 and 33% positive samples respectively). During the second experiment, the EHP-water was subjected to chemical (20 ppm calcium hypochlorite) and to resting with aeration for 5 days and 10 days before addition of naive PL-12 to determine its infectivity. Within 16 days, the PL-12 in untreated EHP-water and in 5-days rested water developed EHP infections (50% and 10%, respectively), while the PL-12 cultured in EHP-water treated with 20 ppm calcium hypochlorite and in 10-days rested water remained uninfected. Altogether results showed that EHP contaminated water is a potential transmission risk but that chemical disinfection or resting for a suitable period in the absence of carriers can substantially reduce its infectivity.