Phylogeny, morphology, virulence, ecology, and host range of Ordospora pajunii (Ordosporidae), a microsporidian symbiont of Daphnia spp.

The impacts of microsporidia on host individuals are frequently subtle and can be context dependent. A key example of the latter comes from a recently discovered microsporidian symbiont of Daphnia, the net impact of which was found to shift from negative to positive based on environmental context. Given this, we hypothesized low baseline virulence of the microsporidian; here, we investigated the impact of infection on hosts in controlled conditions and the absence of other stressors. We also investigated its phylogenetic position, ecology, and host range. The genetic data indicate that the symbiont is Ordospora pajunii, a newly described microsporidian parasite of Daphnia. We show that O. pajunii infection damages the gut, causing infected epithelial cells to lose microvilli and then rupture. The prevalence of this microsporidian could be high (up to 100% in the lab and 77% of adults in the field). Its overall virulence was low in most cases, but some genotypes suffered reduced survival and/or reproduction. Susceptibility and virulence were strongly host-genotype dependent. We found that North American O. pajunii were able to infect multiple Daphnia species, including the European species Daphnia longispina, as well as Ceriodaphnia spp. Given the low, often undetectable virulence of this microsporidian and potentially far-reaching consequences of infections for the host when interacting with other pathogens or food, this Daphnia-O. pajunii symbiosis emerges as a valuable system for studying the mechanisms of context-dependent shifts between mutualism and parasitism, as well as for understanding how symbionts might alter host interactions with resources. IMPORTANCE: The net outcome of symbiosis depends on the costs and benefits to each partner. Those can be context dependent, driving the potential for an interaction to change between parasitism and mutualism. Understanding the baseline fitness impact in an interaction can help us understand those shifts; for an organism that is generally parasitic, it should be easier for it to become a mutualist if its baseline virulence is relatively low. Recently, a microsporidian was found to become beneficial to its Daphnia hosts in certain ecological contexts, but little was known about the symbiont (including its species identity). Here, we identify it as the microsporidium Ordospora pajunii. Despite the parasitic nature of microsporidia, we found O. pajunii to be, at most, mildly virulent; this helps explain why it can shift toward mutualism in certain ecological contexts and helps establish O. pajunii is a valuable model for investigating shifts along the mutualism-parasitism continuum.

Near chromosome-level genome assembly of the microsporidium Hamiltosporidium tvaerminnensis.

Microsporidia are intracellular parasitic fungi whose genomes rank among the smallest of all known eukaryotes. A number of outstanding questions remain concerning the evolution of their large-scale variation in genome architecture, responsible for genome size variation of more than an order of magnitude. This genome report presents the first near-chromosomal assembly of a large-genome microsporidium, Hamiltosporidium tvaerminnensis. Combined Oxford Nanopore, Pacific Biosciences (PacBio), and Illumina sequencing led to a genome assembly of 17 contigs, 11 of which represent complete chromosomes. Our assembly is 21.64 Mb in length, has an N50 of 1.44 Mb, and consists of 39.56% interspersed repeats. We introduce a novel approach in microsporidia, PacBio Iso-Seq, as part of a larger annotation pipeline for obtaining high-quality annotations of 3,573 protein-coding genes. Based on direct evidence from the full-length Iso-Seq transcripts, we present evidence for alternative polyadenylation and variation in splicing efficiency, which are potential regulation mechanisms for gene expression in microsporidia. The generated high-quality genome assembly is a necessary resource for comparative genomics that will help elucidate the evolution of genome architecture in response to intracellular parasitism.

Detection of a novel microsporidium with intranuclear localization in farmed Penaeus vannamei from Latin America.

Microsporidia are emerging intracellular parasites of most known animal phyla in all ecological niches. In shrimp aquaculture, the microsporidium Enterocytozoon hepatopenaei (EHP) is a major cause of concern inflicting tremendous losses to shrimp producers in southeast Asia. During a histopathological examination of Penaeus vannamei samples originating in a country from Latin America presenting slow growth, we observed abnormal nuclei in the epithelial cells of the hepatopancreas. A PCR screening of the samples using DNA isolated from paraffin embedded tissues for the SSU rRNA gene of EHP provided a 149 bp amplicon. In situ hybridization using the SSU rRNA gene probe provided a positive signal in the nuclei instead of the cytoplasm. Sequence analysis of the SSU rRNA gene product revealed a 91.3 %, 89.2 % and 85.4 % sequence identity to Enterocytozoon bieneusi, E. hepatopenaei and Enterospora canceri respectively. Furthermore, phylogenetic analysis revealed the newly discovered microsporidium clustered with E. bieneusi. Considering the intranuclear location of the novel microsporidium and the differences in the sequence of the SSU rRNA, we tentatively consider this parasite a new member of the genus Enterospora sp. The pathogenicity and distribution of the shrimp Enterospora sp. are currently unknown. Our future efforts are focused on the characterization and development of diagnostic tools for this parasite to understand if it acts as an emergent pathogen that might require surveillance to prevent its spread.

Application of PCR-based diagnostic tools that target Enterocytozoon hepatopenaei for the molecular detection of a Vittaforma-like microsporidium that infects Penaeus vannamei from Costa Rica.

Several PCR methodologies are available for the detection of Enterocytozoon hepatopenaei (EHP) that target the SSU rRNA gene. However, these methodologies are reported as unsuitable for the detection of EHP due to specificity issues. Here, we report the applicability of two commonly used SSU rRNA methodologies for the detection of additional microsporidia from the genus Vittaforma that is present in cultured Penaeus vannamei from Costa Rica. The molecular detection of DNA of the novel microsporidia can only be achieved using SSU rRNA targeting methodologies and does not cross-react with the highly specific spore wall protein gene PCR detection method.

Morphological characterization and genetic diversity of a new microsporidium, Neoflabelliforma dubium n. sp. from the adipose tissue of Diaphanosoma dubium (Crustacea: Sididae).

We reported a new microsporidium Neoflabelliforma dubium n. sp. from the adipose tissue of Diaphanosoma dubium in China. The infected daphnids generally appeared opaque due to the presence of numerous spore aggregates located in the adipose tissue. All developmental stages were in direct contact with the host cell cytoplasm. Multinucleate sporogonial plasmodia developed into uninucleate sporoblasts by rosette-like fashion. Mature spores were pyriform and monokaryotic, measuring 4.02 ± 0.24 (3.63-4.53) µm long and 2.27 ± 0.15 (2.12-2.57) µm wide (N = 40). The polaroplast was bipartite with a tightly packed anterior lamellae and a loosely aligned posterior lamellae. Isofilar polar filament was coiled 9-11 turns and arranged in 2-3 rows. The phylogenetic analysis based on the obtained SSU rDNA sequence indicated that the N. dubium n. sp. clustered with the freshwater oligochaete-infecting N. aurantiae to form an independent monophyletic group, positioned at the base of Clade 4. In addition, we analyzed the genetic diversity in three N. dubium n. sp. isolates based on the rDNA (SSU rDNA, ITS and LSU rDNA) and Rpb1 gene. The genetic variation among the rDNA sequences was not distinct, however, high nucleotide diversity could be observed in Rpb1 gene, and a wide variety of Rpb1 haplotypes were identified within each isolate. Genetic recombination detected in the Rpb1 sequences presumes cryptic sexual process occurring in N. dubium n. sp. Statistical evolutionary analyses further indicated that the purifying selection eliminated mutations in the Rpb1 gene.

A new microsporidian parasite, Microsporidium theragrae n. sp., infecting Alaska pollock Gadus chalcogrammus (Teleostei: Gadidae).

A new microsporidian infecting Gadus chalcogrammus Pallas, 1814 (Gadidae), is described based on morphological, ultrastructural, and molecular studies. This microsporidian parasite develops inside intramuscular spindle-shaped lesions measuring approximately 1-2 mm in width and 4-8 mm in length. Infected cells encapsulated by a host-produced wall containing a sponge-like acellular zone. Sporogony presumably proceeds via segmentation of sporogonial plasmodium, resulting in a variable number of spores. Sporogonial stages develop in sporophorous vesicles (SVs), abutting a moderately electron-dense thick walled coat of a homogeneous amorphous material. SVs space contains rare granular and tubular inclusions. Neighboring SVs often interconnected by bridges of the host cell cytoplasm that were limited by membrane comparable with SV coat. The elongate-ovoid spores, measuring 4.29 ± 0.38 × 2.51 ± 0.26 µm (N 104), possess a bipartite polaroplast and polar tube with 15-16 coils arranged in 2-3 layers. The angle of tilt of the polar tube coils is less than 30°. The sequence analysis of SSU rDNA coding region showed that the studied microsporidians differs from other fish muscle-infecting species at least in 17 bp (2.58%) and is closely related to Microsporidium cypselurus Yokoyama et al. (2002) infecting the flying fish from East China Sea. The parasite is provisionally positioned as Microsporidium theragrae sp. n.

First record of a new microsporidium pathogenic to Gonipterus platensis in Brazil.

Microsporidia are naturally occurring fungal-related parasites that can infect nearly all animal hosts, but their biocontrol potential of insect pests is routinely overlooked in agriculture and forestry. This research brings the first report describing the natural occurrence of a microsporidium causing disease in field-collected populations of the invasive eucalyptus snout beetle, Gonipterus platensis (Coleoptera: Curculionidae), a major destructive pest of eucalyptus plantations in Brazil. Adult beetles were collected during field surveys in commercial eucalyptus plantations in southern Brazil to be examined and dissected with typical symptoms to verify presence of microsporidian spores in haemolymph. From 14 plantations in different sites, the natural infection occurrence in these populations ranged from 0 to 65%, while a lab colony exhibited an infection incidence of 70%. Spore density in haemolymph of symptomatic insects averaged 2.1 (± 0.4) × 107 spores/beetle. Symptoms in infected adults were identified by an abnormal abdomen with malformation of the second pair of wings, impairing their flight activity. Electron transmission microscopy of the pathogen showed morphological features similar to species belonging to the genus Nosema or Vairimorpha. Phylogenetic analysis of the full-length small subunit ribosomal RNA gene suggests this pathogen's placement in the genus Vairimorpha, but with a sequence identity of ~ 94% with the nearest neighbours. The low level of sequence identity suggests this pathogen may represent a novel taxon in the genus and further requires whole genome sequencing for definitive taxonomic resolution. These findings provide insights on the natural occurrence of this novel pathogen of this invasive pest in Eucalyptus plantations in Brazil. Further studies are needed to determine potential of this microsporidium in the design of conservative or augmentative biological control programs for this invasive pest.

Tetra disseminated microsporidiosis: a novel disease in ornamental fish caused by Fusasporis stethaprioni n. gen. n. sp.

A novel microsporidial disease was documented in two ornamental fish species, black tetra Gymnocorymbus ternetzi Boulenger 1895 and cardinal tetra Paracheirodon axelrodi Schultz 1956. The non-xenoma-forming microsporidium occurred diffusely in most internal organs and the gill, thus referring to the condition as tetra disseminated microsporidiosis (TDM). The occurrence of TDM in black tetra was associated with chronic mortality in a domestic farmed population, while the case in cardinal tetra occurred in moribund fish while in quarantine at a public aquarium. Histology showed that coelomic visceral organs were frequently necrotic and severely disrupted by extensive infiltrates of macrophages. Infected macrophages were presumed responsible for the dissemination of spores throughout the body. Ultrastructural characteristics of the parasite developmental cycle included uninucleate meronts directly in the host cell cytoplasm. Sporonts were bi-nucleated as a result of karyokinesis and a parasite-produced sporophorous vesicle (SPV) became apparent at this stage. Cytokinesis resulted in two spores forming within each SPV. Spores were uniform in size, measuring about 3.9 ± 0.33 long by 2.0 ± 0.2 µm wide. Ultrastructure demonstrated two spore types, one with 9-12 polar filament coils and a double-layered exospore and a second type with 4-7 polar filament coils and a homogenously electron-dense exospore, with differences perhaps related to parasite transmission mechanisms. The 16S rDNA sequences showed closest identity to the genus Glugea (≈ 92%), though the developmental cycle, specifically being a non-xenoma-forming species and having two spores forming within a SPV, did not fit within the genus. Based on combined phylogenetic and ultrastructural characteristics, a new genus (Fusasporis) is proposed, with F. stethaprioni n. gen. n. sp. as the type species.

Molecular identification of microsporidian species in patients with epithelial keratitis.

Introduction. Ocular microsporidiosis is a significant emerging infectious disease reported in immunocompromised patients and immunocompetent persons throughout the world.Aim. To identify the pathogens responsible for human keratitis, via corneal scrapings.Methodology. Thirty-three hospitalized patients with epithelial keratitis were examined using staining and DNA sequencing. DNA was extracted from corneal samples and the small-subunit ribosomal RNA gene was amplified by polymerase chain reaction (PCR) and sequenced.Results. Twenty-one samples were positive by staining while PCR generated amplicons in 18 cases. Of the 18 sequences, 16 were identical with, or very similar to, those of Vittaforma corneae (99-100 % similarity) and the remaining two sequences were similar to that of unidentified Microsporidium species deposited in the GenBank.Conclusion. This study has reconfirmed that V. corneae causes epithelial keratitis in humans and that a newly detected Microsporidium species is also involved in microsporidial keratitis as one of the emerging pathogens in Thailand. Ophthalomologists should be aware of microsporidial keratitis in people from Thailand and those from neighbouring countries.

Ultrastructure, molecular phylogeny, and prevalence rates of Alternosema bostrichidis gen. nov. sp. nov. (Microsporidia, Terresporidia), a parasite of Prostephanus truncatus and Dinoderus spp. (Coleoptera, Bostrichidae).

A new species and a new genus of a microsporidium Alternosema bostrichidis isolated from an adult Prostephanus truncatus in Mexico and from three species of the genus Dinoderus in Nigeria are described. The microsporidium is monomorphic, monoxenic, and develops in direct contact with host cell cytoplasm. The infection first appears with thoracic muscles, followed by a generalized invasion of the host. All developmental stages are diplokaryotic. Sporogony is disporoblastic. Mature spores are ovoid. Unfixed spores measure 3.7-4.2 × 2.0-2.6 µm, fixed and stained spores 3.5-5.0 × 2.4-2.8 µm. The polaroplast consists of dense lamellae and rare lamellae. The polar tube is slightly anisofilar, consisting of 11-17 coils, with 9-14 proximal (130 nm in diameter) and 2-3 distal coils (120 nm in diameter) arranged in one layer. Molecular phylogenetic analysis based upon a short portion of small-subunit ribosomal RNA gene (Genbank accession # KP455651) placed the new microsporidium within Liebermannia-Orthosomella lineage, which contains multiple undescribed parasites. In particular, A. bostrichidis showed maximal sequence similarity of 95% to Microsporidium sp. BBRE2 (# FJ755987) from Baikalian Diplacanthus brevispinus (Amphipoda: Acanthogammaridae) and Microsporidium sp. Comp CD Van 2 (# KC111784) from compost and soil in Canada. Frequent, devastating epizootics of laboratory cultures of A. bostrichidis support its potential as a biological control agent of grain borers.

A Vavraia-like microsporidium as the cause of deadly infection in threatened and endangered Eurycea salamanders in the United States.

BACKGROUND: Eurycea sosorum (Barton Springs salamander) and Eurycea nana (San Macros salamander) are listed as endangered and threatened species, respectively, by the U.S. Fish and Wildlife Service (USFWS) with habitats restricted to small regions near Austin, Texas, USA. The conservation efforts with the Eurycea salamanders at the captive breeding program in San Marcos Aquatic Resources Center (SMARC), a USFWS facility, have seen an unexpected and increased mortality rate over the past few years. The clinical signs of sick or dead salamanders included erythema, tail loss, asymmetric gills or brachial loss, rhabdomyolysis, kyphosis, and behavior changes, suggesting that an infectious disease might be the culprit. This study aimed to identify the cause of the infection, determine the taxonomic position of the pathogen, and investigate the potential reservoirs of the pathogen in the environment. RESULTS: Histopathological examination indicated microsporidian infection (microsporidiosis) in the sick and dead Eurycea salamanders that was later confirmed by PCR detection. We also determined the near full-length small subunit ribosomal RNA (SSU rRNA) gene from the microsporidian pathogen, which allowed us to determine its phylogenetic position, and to design primers for specific and sensitive detection of the pathogen. Phylogenetic analysis indicated that this pathogen was closely related to the insect parasites Vavraia spp. and the human opportunistic pathogen, Trachipleistophora hominis. This Vavraia-like microsporidium was present in dead salamanders at SMARC archived between 2011 and 2015 (positive rates ranging between 52.0-88.9% by PCR detection), as well as in some aquatic invertebrates at the facility (e.g. snails and small crustaceans). CONCLUSIONS: A Vavraia-like microsporidian was at least one of the major pathogens, if not solely, responsible for the sickness and mortality in the SMARC salamanders, and the pathogen had been present in the center for years. Environmental invertebrates likely served as a source and reservoir of the microsporidian pathogen. These observations provide new knowledge and a foundation for future conservation efforts for Eurycea salamanders including molecular surveys, monitoring of the pathogen, and discovery of effective treatments.

Identification and localization of SAS-6 in the microsporidium Nosema bombycis.

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

Histopathological, ultrastructural and molecular phylogenetic analysis of a novel microsporidium in a loggerhead sea turtle Caretta caretta.

Microsporidial spores were identified in the musculature of a loggerhead sea turtle Caretta caretta found dead on the shore in New Brunswick, Canada. Gastroenteritis was diagnosed on gross postmortem examination, with no gross abnormalities detected in the skeletal muscle. Histologically, the microsporidial spores were associated with inflammation and muscular necrosis and measured 1.1-1.7 × 2.2-3.4 µm. Spores were typically identified within sporophorous vesicles and, less often, in sporophorocysts and were weakly Gram positive, had punctate PAS staining, and were occasionally strongly acid-fast. Ultrastructural characteristics included 7-10 polar filament coils and other standard features of microsporidial spores. PCR for the microsporidial small subunit rRNA gene sequence was performed on DNA extracted from the muscle and small intestine, and the resulting amplicon was sequenced and queried against published microsporidial genomes. DNA sequences shared 98.2-99.8% sequence identity to Clade III of the Marinosporidia. This is the first report of a microsporidial infection contributing to the mortality of a sea turtle.

Discovery of a novel microsporidium in laboratory colonies of Mediterranean cricket Gryllus bimaculatus (Orthoptera: Gryllidae): Microsporidium grylli sp. nov.

A microsporidium was found in a Mediterranean cricket Gryllus bimaculatus from a pet market in the UK and a lab stock at the Moscow Zoo (originating from London Zoo). The spores were ovoid, uninucleate, 6.3 × 3.7 µm in size (unfixed), in packets by of 8, 16, or 32. The spores were easily discharged upon dessication or slight mechanical pressure. The polar tube was isofilar, with 15-16 coils arranged in 1-2 rows. The polaroplast was composed of thin lamellae and occupied about one third of the spore volume. The endospore was 200 nm thick, thinning over the anchoring disc. The exospore was thin, uniform, and with no ornamentation. Phylogenetics based upon small subunit ribosomal RNA (Genbank accession # MG663123) and RNA polymerase II largest subunit (# MG664544) genes placed the parasite at the base of the Trachipleistophora/Vavraia lineage. The RPB1 locus was polymorphic but similar genetic structure and identical clones were found in both isolates, confirming their common geographic origin. Due to in insufficient ultrastructural data and prominent divergence from described species, the parasite is provisionally placed to the collective taxon Microsporidium.

The role of spore morphology in horizontal transmission of a microsporidium of Daphnia.

The microsporidian parasite Hamiltosporidium tvaerminnensis can infect Daphnia magna both horizontally (through environmental spores) and vertically (through parthenogenetic and sexually produced eggs). The spores of H. tvaerminnensis come in three distinguishable morphologies, which are thought to have different roles in the transmission of the parasite. In this study, we examined the role of the two most common spore morphologies (i.e. oval-shaped spores and pear-shaped spores) in horizontal transmission of H. tvaerminnensis. To this end, we infected hosts with solutions consisting of either mostly oval- or mostly pear-shaped spores, and quantified infection rates, parasite-induced host mortality and mean number of parasite spores produced per host. We found that spore morphology by itself did not influence infection rates and parasite-induced host mortality. Instead, host clone and parasite isolate interacted with spore morphology in shaping infection outcome and mortality. Thus, there appear to be strong genotype-by-genotype (G × G) interactions in this system. While there is no dispute that H. tvaerminnensis can transmit both vertically and horizontally, our findings do not support theoretical predictions that different spore morphologies hold different roles in horizontal transmission of H. tvaerminnensis.

Description and phylogeny of a new microsporidium from the elm leaf beetle, Xanthogaleruca luteola Muller, 1766 (Coleoptera: Chrysomelidae).

This study describes a new genus and species of microsporidia which is a pathogen of the elm leaf beetle, Xanthogaleruca luteola Muller, 1776 (Coleoptera: Chrysomelidae). The beetles were collected from Istanbul in Turkey. All developmental stages are uninucleate and in direct contact with the host cell cytoplasm. Giemsa-stained mature spores are oval in shape and measured 3.40 ± 0.37 µm in length and 1.63 ± 0.20 µm in width. These uninucleate spores have an isofilar polar filament with 11 turns. The spore wall was trilaminar (75 to 115 nm) with a rugose, electron-dense exospore (34 to 45 nm) and a thickened, electron-lucent endospore (65 to 80 nm) overlaying the plasmalemma. Morphological, ultrastructural, and molecular features indicate that the described microsporidium is dissimilar to all known microsporidian taxa and confirm that it has different taxonomic characters than other microsporidia infecting X. luteola and is named here as Rugispora istanbulensis n. gen., n. sp.

Parahepatospora carcini n. gen., n. sp., a parasite of invasive Carcinus maenas with intermediate features of sporogony between the Enterocytozoon clade and other microsporidia.

Parahepatospora carcini n. gen. n. sp., is a novel microsporidian parasite discovered infecting the cytoplasm of epithelial cells of the hepatopancreas of a single Carcinus maenas specimen. The crab was sampled from within its invasive range in Atlantic Canada (Nova Scotia). Histopathology and transmission electron microscopy were used to show the development of the parasite within a simple interfacial membrane, culminating in the formation of unikaryotic spores with 5-6 turns of an isofilar polar filament. Formation of a multinucleate meront (>12 nuclei observed) preceded thickening and invagination of the plasmodial membrane, and in many cases, formation of spore extrusion precursors (polar filaments, anchoring disk) prior to complete separation of pre-sporoblasts from the sporogonial plasmodium. This developmental feature is intermediate between the Enterocytozoonidae (formation of spore extrusion precursors within the sporont plasmodium) and all other Microsporidia (formation of spore extrusion precursors after separation of sporont from the sporont plasmodium). SSU rRNA-based gene phylogenies place P. carcini within microsporidian Clade IV, between the Enterocytozoonidae and the so-called Enterocytospora-clade, which includes Enterocytospora artemiae and Globulispora mitoportans. Both of these groups contain gut-infecting microsporidians of aquatic invertebrates, fish and humans. According to morphological and phylogenetic characters, we propose that P. carcini occupies a basal position to the Enterocytozoonidae. We discuss the discovery of this parasite from a taxonomic perspective and consider its origins and presence within a high profile invasive host on the Atlantic Canadian coastline.

Occurrence of Microsporidium sp. and other pathogens in Ips amitinus (Coleoptera: Curculionidae).

A new microsporidium is reported from the small spruce bark beetle, Ips amitinus: Microsporidium sp. with uninucleate oval spores measuring 3.5 × 2.5 µm; infecting cells of the midgut epithelium, midgut muscles, the fat body, the Malpighian tubules, and the gonads of adult beetles collected in Austria. Seven other pathogens were found in beetles collected from Austria, the Czech Republic, and Finland. Six of them were already known from I. amitinus. Nosema cf. typographi is recorded for the first time in the overwintering generation of I. amitinus from the Czech Republic.

Molecular identification of Nucleophaga terricolae sp. nov. (Rozellomycota), and new insights on the origin of the Microsporidia.

Microsporidia are widespread endoparasites of animals, including humans. They are characterized by highly modified morphological and genetic features that cause difficulties in elucidating their enigmatic origin and evolution. Recent advances, however, indicate that the Microsporidia have emerged from the Rozellomycota, forming together either the most basal lineage of the Fungi or its closer relative. The Rozellomycota comprise a huge diversity of uncultured environmental clones, with a very few known species endoparasitic of algae and water moulds, like the chytrid-like Rozella, and of free-living amoebae, like Nucleophaga and the microsporidia-like Paramicrosporidium. A possible ancestral microsporidium, Mitosporidium, has recently been described from the water flea Daphnia, since the phylogenomic reconstruction showed that it branches to the root of the microsporidian tree, while the genome analysis revealed a fungal-like nuclear genome and the persistence of a mitochondrial genome. Here we report the 18S rDNA molecular phylogeny of an additional microsporidium-like endoparasite of amoebae, which has a developmental cycle almost identical to that of Nucleophaga amoebae. Our results show that the endoparasite is closely related to N. amoebae, forming a distinct species, for which we propose the name Nucleophaga terricolae. Furthermore, the Nucleophaga lineage is recovered as sister to the Microsporidia while Mitosporidium turns out to be member of a well-supported group of environmental clones. These results raise the question about the actual ancestry of the Microsporidia within the Rozellomycota. A precise and robust phylogeny will require further comparative genomic studies of these various strains, and should also consider the primitive microsporidia, for which genetic data are still lacking, because all these organisms are essentially morphologically similar.

A new microsporidium, Potaspora macrobrachium n.sp. infecting the musculature of pond-reared oriental river prawn Macrobrachium nipponense (Decapoda: Palaemonidae).

This paper described a novel microsporidian infection in the pond-reared oriental river prawn Macrobrachium nipponense. A conspicuous symptom of the infection was progressive white opacity associated with the musculature. Although neither bacteria nor viruses were detected in routine diagnostic tests, apparently degenerated microsporidian cells or spores were frequently observed in wet smears of the musculature from diseased prawns. Histological observations also revealed characteristics typical of microsporidian infection throughout the host. Transmission electron microscopy revealed multiple life stages of a microsporidian parasite within the cytoplasm of host muscle cells. In addition, partial small subunit ribosomal RNA (SSU rRNA) gene was obtained by a nested PCR using microsporidian specific primers. A consensus sequence was then deposited in GenBank (accession no. KU307278) and subjected to a general BLASTn search that yielded hits only for microsporidian sequence records. Phylogenetic analysis showed that the isolate was most similar to the fish microsporidian clade containing the genera Kabatana, Microgemma, Potaspora, Spraguea, and Teramicra. The highest sequence identity, 87%, was with Potaspora spp. Based on histological, ultrastructure and molecular phylogenetic data, we erected a new species, Potaspora macrobrachium for the novel microsporidium. The description of microsporidium in this important commercial host was fundamental for future consideration of factors affecting stock health and sustainability.