Sporogonial Plasmodia Research Articles

Host and transmission route of Enterocytozoon hepatopenaei (EHP) between dragonfly and shrimp

Enterocytozoon hepatopenaei (EHP) is a shrimp pathogen that causes huge economic losses. In the present study, the hosts of EHP were investigated using polymerase chain reaction (PCR) in an aquaculture farm located in Maoming, China. EHP was detected in Litopenaeus vannamei, Penaeus monodon, Varuna litterata, Mytilopsis leucophaeata, and three dragonfly species (Anax parthenope, Pantala flavescens, and Ischnura senegalensis). In the histopathological examination using hematoxylin–eosin (HE) staining, EHP spores were found in nymphs and adult dragonflies naturally infected with EHP that were collected from the shrimp farm. Fluorescence in situ hybridization (FISH) results showed a positive signal for EHP infection in the fat body of dragonfly nymphs. Immature and mature microsporidian spores and late sporogonial plasmodium were observed in the cytoplasm of dragonfly nymphs using transmission electron microscopy (TEM). The transmission of EHP from shrimp to dragonfly nymphs was confirmed via cohabitation challenge experiments in which EHP-free dragonfly nymphs were cohabited with EHP-infected shrimp, and the transmission of EHP from dragonfly nymphs to shrimp was demonstrated via the cohabitation of EHP-infected dragonfly nymphs with EHP-free shrimp and oral administration challenge experiments. This study confirms that dragonflies can act as natural EHP hosts, and a novel EHP horizontal transmission route exists between dragonflies and shrimp.

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 1-2mm in width and 4-8mm 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 ± 0.26μm (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 17bp (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.

Naidispora caidianensis n. gen. n. sp. infecting coelomocytes of oligochaete Branchiura sowerbyi (Oligochaeta: Naididae) in China

A new microsporidian parasite, Naidispora caidianensis n. gen. n. sp. was found in coelomocytes of oligochaete Branchiura sowerbyi Beddard, 1892 from Wuhan city, Hubei Province, China. Opaque, hypertrophied coelomocytes (0.17–0.24 mm in diameter) depicted clinical signs of infection. Electron microscopy revealed a microsporidian with monokaryotic life stages. Rounded uninucleate meronts subsequently transformed into multinucleate merogonial plasmodia with masses of electron-dense projections on their plasma membrane. Electron–dense sporogonial plasmodia separated into uninucleate sporonts through rosette-like budding, and further developed into sporoblasts, enclosed by a sporophorous vesicle. Uninucleate mature spores were pyriform, measured 4.1 ± 0.1 (3.9–4.3) μm × 2.1 ± 0.07 (1.9–2.2) μm (average ± SE, range, n = 50), and contained a mushroom-like anchoring disk, bipartite polaroplast, electron-lucent posterior vacuole, trilaminar spore wall and 12–14 turns of an isofilar polar filament arranged in 2–3 ranks. The 1542 bp sequence from B. soweryi showed the highest similarity (below 80 %) with Nematocenator marisprofundi (accession number: JX463178). SSU rRNA gene-based phylogenetic analysis demonstrated that the novel taxon formed an independent clade from known microsporidian parasites. Based on the ultrastructural features and SSU rRNA gene sequence, we propose the establishment of a novel genus (Naidispora n. gen.) and species (Naidispora caidianensis n. sp.) to contain this parasite.

Nucleospora hippocampi n. sp., an Intranuclear Microsporidian Infecting the Seahorse Hippocampus erectus From China

The life cycle, ultrastructure, and molecular phylogeny of a new intranuclear microsporidian, Nucleospora hippocampi n. sp., infecting the intestine of the Hippocampus erectus, were described. The histopathology revealed an extensive infection, mainly in the columnar epithelium of the intestinal mucosa layer. The enterocytes were the important target cell for Nucleospora hippocampi n. sp. infection. Transmission electron microscopy results showed that this microsporidian developed directly within the host cell nucleoplasm. In the intranuclear life cycle, the transformation from meront to sporogonial plasmodium was recognized by forming electron-dense disc structures, which were considered the polar tube precursors. The microsporidian showed the typical morphological characteristics of the family Enterocytozoonidae in the formation and development of spore organelles prior to the division of the sporogonial plasmodium. According to wet smear observation, eight spores were generally formed in a single host nucleus. Mature spores were elongated ovoids that were slightly bent and measured 1.93 × 0.97 μm. The isofilar polar tube was arranged in 7~8 coils in one row. Phylogenetic analysis of its small subunit ribosomal DNA sequences demonstrated that the parasite belonged to the Nucleospora group clade. The histological, ultrastructural, and molecular data support the emergence of a new species in the genus Nucleospora. This is the first report of Nucleospora species in Asia and threatened syngnathid fishes.

More about the biodiversity of parasites in Vietnam: a first report of microsporidia (opistosporidia, microsporidia) in coral fish

This is the first report of microsporidia infection in marine fish from Vietnam. Microsporidia (Opistosporidia, Microsporidia) were identified in 4 species of coral fish Cheilinus chlorourus (Labridae), Abudefduf bengalensis (Pomacentridae), Pomacentrus brachialis (Pomacentridae), Atule mate (Carangidae) from Nha Trang Bay (East Sea), Khanh Hoa Province, Vietnam, in 2018 and 2019. Dry smears of blood, intestine and liver were stained using the Giemsa method. Gut and liver were fixed in Bouin’s fluid, processed for routine paraffin infiltration, and sections were stained with Hematoxylin and Eosin. Strong microsporidia infections were the same for all three species revealed in 1 of every 6 fish samples (prevalence 16.7%) of C. chlorourus, A. bengalensis, and P. brachialis. In C. chlorourus there were meronts, sporogonial plasmodia, and sporoblasts found in all layers of intestine (enterocytes, myocytes, mesotheliocytes), pancreas and liver. In A. bengalensis parasites were found in enterocytes of the small intestine and liver. In P. brachialis meronts, sporogonial plasmodia and sporoblasts were in enterocytes of the small intestine. Intracellular parasites are located in parasitophorous vacuoles covered with additional shell, which appeared to be in the process of forming xenoma. Microsporidia infection was revealed in 1 of 31 fish samples of A. mate (prevalence 3 %). Up to 30 of round and oval white xenomas were detected in the gills of fresh samples by microscopy. We tentatively assign the microsporidia to Glugeidae due to host affiliation (fish), localization (digestive system) and xenoma formation. They differ from Pleistophoridae by localization in non-muscular cells.

Morphological and phylogenetic characterization of a new microsporidium, Triwangia gracilipes n. sp. From the freshwater shrimp Caridina gracilipes (Decapoda: Atyidae) in China

A new microsporidian species was described from the freshwater shrimp Caridina gracilipes collected from Lake Luoma located in Northern Jiangsu province, East China. The infected shrimps appeared generally opaque due to the presence of white cysts located in the connective tissues of the surface of the hepatopancreas. The earliest developmental stages observed were diplokaryotic meronts which were in direct contact with the host cell cytoplasm. Multinucleate sporogonial plasmodia developed into uninucleate sporoblasts which were enclosed in sporophorous vesicles. The parasite developed synchronously within an individual sporophorous vesicle. Mature spores were pyriform and monokaryotic, measuring 5.45 ± 0.18 (5.12–5.82) µm long and 3.57 ± 0.17 (3.18–3.92) µm wide. Anisofilar polar filaments coiled 10–12 turns and arranged in one row. Phylogenetic analysis based on the obtained SSU rDNA sequence indicated that the present species clustered with Triwangia caridina with high support value to form an independent branch which was placed at the basal position of a large clade of containing microsporidia of fishes, crustaceans and amphipods. Based on the morphological characters and ultrastructural features, as well as SSU rDNA-inferred phylogenetic relationships, a new species was erected and named as Triwangia gracilipes n. sp. The taxonomic affiliation of Triwangia was also primarily explored.

Morphological and molecular characterization of a new microsporidium, Janacekia tainanus n. sp. from the adipose tissue of Kiefferulus tainanus (Diptera: Chironomidae) in China

We reported a new microsporidium Janacekia tainanus n. sp. from the adipose tissue of the midge Kiefferulus tainanus Kieffer, 1912 collected from a eutrophic pond in Daye city, Hubei Province, China. Infected chironomid larvae with hypertrophied adipose tissue exhibited porcelain-white. All developmental stages possessed large nuclei. The earliest stages observed were diplokaryotic meronts which were in direct contact with the host adipocyte cytoplasm. Diplokaryotic meronts developed into sporonts with the deposition of electron-dense coagulum on their surface. Multinucleate sporogonial plasmodia developed into uninucleate sporoblasts by the rosette-like division. Mature spores were oval and monokaryotic, measuring 6.14 ± 0.27 (5.65–6.67) µm long and 3.71 ± 0.12 (3.43–3.98) µm wide. Bipartite polaroplast consisted of a narrow anterior lamella and a wide posterior lamella. Isofilar polar filaments coiled 13–17 turns and arranged in one row. The exospore was thin and of no stratification, but remarkably covered with tubular secretions. The electron-lucent endospore was thick and measured 145–352 nm wide. Phylogenetic analysis based on the obtained SSU rDNA sequence indicated that the present species clustered closely with Jirovecia sinensis, a species with rod-shaped mature spores isolated from the coelomocytes of Branchiura sowerbyi. Consistent with the previous result, the monophyletic clade of Jirovecia-Bacillidium-Janacekia was sister to Pseudonosema clade and then collectively nested within Clade V of Class Aquasporidia sensu Vossbrinck and Debrunner-Vossbrinck (2005). The novel species did not form an independent monophyletic lineage with the congener, Janacekia debaisieuxi. Based on the morphological characters and ultrastructural features, as well as SSU rDNA-inferred phylogenetic relationships, a new species in the genus Janacekia, Janacekia tainanus n. sp. was designated. This is the first report of aquatic arthropod-infecting microsporidia in China.

Long rDNA amplicon sequencing of insect-infecting nephridiophagids reveals their affiliation to the Chytridiomycota and a potential to switch between hosts

Nephridiophagids are unicellular eukaryotes that parasitize the Malpighian tubules of numerous insects. Their life cycle comprises multinucleate vegetative plasmodia that divide into oligonucleate and uninucleate cells, and sporogonial plasmodia that form uninucleate spores. Nephridiophagids are poor in morphological characteristics, and although they have been tentatively identified as early-branching fungi based on the SSU rRNA gene sequences of three species, their exact position within the fungal tree of live remained unclear. In this study, we describe two new species of nephridiophagids (Nephridiophaga postici and Nephridiophaga javanicae) from cockroaches. Using long-read sequencing of the nearly complete rDNA operon of numerous further species obtained from cockroaches and earwigs to improve the resolution of the phylogenetic analysis, we found a robust affiliation of nephridiophagids with the Chytridiomycota—a group of zoosporic fungi that comprises parasites of diverse host taxa, such as microphytes, plants, and amphibians. The presence of the same nephridiophagid species in two only distantly related cockroaches indicates that their host specificity is not as strict as generally assumed.

Morphological and molecular characterization of a new species, Agglomerata daphniae n. sp. from the hypoderm of Daphnia magna (Crustacea: Daphniidae)

A new microsporidian species was described from the hypoderm of Daphnia magna sampled from gibel carp (Carassius auratus gibelio) ponds located in Wuhan city, China. The infected cladocerans generally appeared opaque due to numerous plasmodia distributed in the host integument. The earliest stages observed were uninucleate meronts that were in direct contact with the host cell cytoplasm. Meronts developed into multinucleate sporogonial plasmodia enclosed in sporophorous vesicles. Sporoblasts were produced by the rosette-like division of sporogonial division. Mature spores were pyriform and monokaryotic, measuring 4.48 ± 0.09 (4.34–4.65) µm long and 2.40 ± 0.08 (2.18–2.54) µm wide. The polaroplast was bipartite with loose anterior lamellae and tight posterior lamellae. Polar filaments, arranged in two rows, were anisofilar with two wider anterior coils, and five narrower posterior coils. The exospore was covered with fibrous secretions and was composed of four layers. Phylogenetic analysis based on the obtained SSU rDNA sequence, indicated that the present species clustered with three unidentified Daphnia pulicaria-infecting microsporidia with high support values to form a monophyletic lineage, rather than with the congener, Agglomerata cladocera. The barcode motif of the internal transcribed spacer (ITS) region of the novel species was unique among representatives of the “Agglomeratidae” sensu clade (Vávra et al., 2018). Based on the morphological characters and SSU rDNA-inferred phylogenetic analyses, a new species was erected and named as Agglomerata daphniae n. sp. This is the first report of zooplankton-infecting microsporidia in China.

The first case of microsporidiosis in Paramecium.

A new microsporidian species, Globosporidium paramecii gen. nov., sp. nov., from Paramecium primaurelia is described on the basis of morphology, fine structure, and SSU rRNA gene sequence. This is the first case of microsporidiosis in Paramecium reported 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 ± 0.2 μm). 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 the Paramecium aurelia species complex. All susceptible strains belong to closely related P. primaurelia and P. pentaurelia species. Phylogenetic analysis placed the new species in the Clade 4 of Microsporidia and revealed its close relationship to Euplotespora binucleata (a microsporidium from the ciliate Euplotes woodruffi), to Helmichia lacustris and Mrazekia macrocyclopis, microsporidia from aquatic invertebrates.

Detection of the intranuclear microsporidian Enterospora nucleophila in gilthead sea bream by in situ hybridization.

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms.

Amblyospora khaliulini (Microsporidia: Amblyosporidae): Investigations on its life cycle and ecology in Aedes communis (Diptera: Culicidae) and Acanthocyclops vernalis (Copepoda: Cyclopidae) with redescription of the species.

A multi-year study was conducted to examine the natural ecology of the microsporidium Amblyospora khaliulini and more fully characterize parasite development and histopathology in all stages of its primary mosquito host, Aedes communis and intermediate copepod host, Acanthocyclops vernalis with redescription of the species. A. khaliulini exhibits polymorphic development, produces three morphologically and functionally distinct spores, and is both horizontally and vertically transmitted. Development in A. vernalis is restricted to females, occurs within the ovaries and results in death of the host. Development is haplophasic with division by binary and multiple fission producing rosette-shaped sporogonial plasmodia and conical uninucleate spores that are orally infectious to Ae. communis larvae. Both sexes are equally susceptible and infections are confined to testes in males and ovaries in females. Initial stages of development include uninucleate schizonts that undergo karyokinesis forming diplokaryotic meronts that divide repeatedly by binary fission. Sporogony occurs in both host sexes, but sporogenesis does not progress normally in adult males and elliptical, thin walled binucleate spores that function in vertical transmission of the microsporidium via infection of the ovaries and eggs are formed in adult females only. Development of vertically acquired infections in larval Ae. communis hosts occurs within fat body tissue, leads to the production of meiospores in male hosts only and results in death during the 4th larval stadium. Initial development is characterized by merogonial multiplication of diplokarya by synchronous binary division producing additional diplokarya. The cessation of merogony and the onset of sporogony are characterized by the simultaneous secretion of a sporophorous vesicle and meiotic division of diplokarya resulting in the formation of octonucleate sporonts that undergo cytokinesis and sporogenesis to form eight uninucleate, broadly ovoid meiospores enclosed within a sporophorous vesicle. The natural prevalence of patent vertically acquired fat body infections in field populations of Ae. communis ranged from 1.6% to 3.6%. Yearly infection rates in A. vernalis copepods ranged from 57.1% to 15.0%. Prevalence rates of horizontally acquired infections in emerging adult Ae. communis ranged from 69.0% to 11.9% in males and 50.0% to 16.4% in females.

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.

Description of Metchnikovella spiralis sp. n. (Microsporidia: Metchnikovellidae), with notes on the ultrastructure of metchnikovellids.

The present paper reports results of a transmission electron microscopy study of a new metchikovellid microsporidium. It was isolated from gregarines Polyrhabdina sp. inhabiting guts of polychaetes Pygospio elegans sampled at the White Sea silt littoral zone. Free sporogony (FS) occurred in the life cycle of the microsporidium alongside sac-bound sporogony (BS). Free spores resided in a parasitophorous vacuole and were of typical metchnikovellidean structure, uninucleate and oblong. They measured on sections 2·0-3·2×1·3-1·9 μm. The life cycle included pre-sporogonial stages represented by dikaryotic cells and 4-nucleate cells with coupled nuclei. A multinucleate sporogonial plasmodium of FS split in numerous (>10) sporoblasts. In BS segregation of sporoblasts occurred within thick-walled cysts by internal budding. Spore sacs of this microsporidium, measuring on average 11·6×4·7 μm, were limited by a thick electron-dense wall, externally ornamented with spirally wound cords of dense material. These oval spore sacs contained eight barrel-shaped spores, comparable in size and ultrastructure to FS spores. Ultrastructure of both types of spores and intracellular development of the new microsporidium and Metchnikovella spp. were similar, suggesting they belong to the same genus. In this paper we describe a new species Metchnikovella spiralis and discuss morphology of metchnikovellids in the context of putative evolutionary history of Microsporidia.

Morphological and molecular characterization of a microsporidian parasite, Takaokaspora nipponicus n. gen., n. sp. from the invasive rock pool mosquito, Ochlerotatus japonicus japonicus

A new genus and species of Microsporidia, Takaokaspora nipponicus n. gen., n. sp. is described from Ochlerotatus japonicus japonicus (Theobald) and Ochlerotatus hatorii (Yamada) based on light microscope and ultrastructural morphology, developmental features, transmission cycles and comparative sequence analyses of the small subunit ribosomal DNA (SSU rDNA). The microsporidium is both vertically and horizontally transmitted, exhibits dimorphic development alternating between diplokaryotic and monokaryotic stages and produces two morphologically distinct spores, one in larvae and another in adult females. Horizontal transmission of infection to larval mosquitoes occurs via direct oral ingestion of uninucleate spores that are produced in vertically-infected larval hosts. Development in horizontally-infected hosts is diplokaryotic following karyokinesis of uninucleate schizonts and binary fission to produce small (4.3μm×2.0μm) membrane free, ovoid, binucleate spores that are confined to adult female reproductive tissues (ovariole sheath and oviducts). Vertical transmission of the microsporidium from adult females to larval progeny takes place via surface contamination of the egg (transovum). Microsporidian development in vertically-infected larvae is haplophasic with unpaired nuclei throughout, producing rosette-shaped sporogonial plasmodia contained within a thin non-persistent sporophorous vesicle and culminating in the formation of membrane free, uninucleate, conical spores (7.0μm×2.8μm). Development is confined to host fat body tissue which appears as swollen white masses in the thorax and selected segments of the abdomen causing larvae to appear abnormally distorted and results in death during the third and fourth instar stages. The SSU rDNA sequences obtained from the two morphologically identical microsporidia isolated from Oc. j. japonicus and Oc. hatorii were nearly identical and unique when compared with GenBank entries of all other mosquito-parasitic species. Phylogenetic trees constructed by Maximum Parsimony, Maximum Likelihood and bootstrap analyses using the Neighbor Joining search parameter yielded similar typologies. In each case, the novel microsporidium was the sister group to the clade containing Parathelohania species from Anopheles mosquitoes and the monotypic Novothelohania ovalae from Ochlerotatus caspius showing approximately 10–13% sequence divergence to those two genera providing strong support for establishment as a separate genus.

Fine structure of Metchnikovella incurvata Caullery and Mesnil 1914 (microsporidia), a hyperparasite of gregarines Polyrhabdina sp. from the polychaete Pygospio elegans

Class Rudimicrosporea Sprague 1977, with its single family Metchnikovellidae, comprises hyperparasites of gregarines from the guts of marine invertebrates. Metchnikovellids remain poorly studied in spite of their significance to the evolutionary history of microsporidia; their ultrastructure and life cycles require further investigation. Here we present results of the light- and electron-microscopy study of Metchnikovella incurvata Caulleri and Mesnil 1914, isolated from lecudinid gregarines, parasitizing polychaetes Pygospio elegans in the White Sea littoral zone, and yet described only on the light-microscopic level. The life cycle of this microsporidium includes 2 sporogonies: free (FS) and sac-bound (SBS). In FS, sporonts develop into multinuclear cells (sporogonial plasmodia), which generate sporoblasts and free spores residing in direct contact with the host cytoplasm. Electron microscopy revealed their metchnikovellidean structure: a horseshoe-shaped nucleus, short manubrium perpendicular to the long axis of the spore, and a polar cap in a separate membrane container. Merogony was not observed. The earliest stages of SBS were chains of binucleate cells. They underwent a series of nuclear and cell divisions, produced extracellular envelopes, and split into boomerang-shaped spore sacs, containing up to 16 spores each. Ultrastructure and sizes of sac-bounded spores were similar to those of free-living ones. An amended diagnosis of M. incurvata is provided.

Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. infecting invasive Chinese mitten crabs (Eriocheir sinensis) in Europe

We describe a microsporidian parasite infecting non-native Chinese mitten crabs (Eriochier sinensis) from Europe. Electron microscopy revealed merogonic and sporogonic life stages bound within a plasmalemma. The crab parasite develops polar tube precursors at the sporont stage but does not complete formation of the intact spore extrusion apparatus at the stage of the sporogonial plasmodium like Enterocytozoon bienuesi and other representatives of the Enterocytozoonidae. Its presence within an aquatic crustacean host, and a distinct molecular phylogeny based on partial small subunit ribosomal RNA (SSU rRNA) gene sequences also place it relatively close, though distinct to, existing genera within the Enterocytozoonidae. Consideration of morphological and phylogenetic characteristics of other hepatopancreas-infecting microsporidia from crustaceans suggests that certain ones (e.g. Enterospora canceri) are retained within the clade corresponding to the existing family Enterocytozoonidae, while others, including the parasite described here, may eventually be grouped in a sister taxon potentially of family rank. Based upon morphological and host similarity, it is likely that the parasite described here is the same as Endoreticulatus eriocheir (Wang and Chen, 2007), previously described from Chinese mitten crabs in Asia. However, using a combined taxonomic approach based upon morphological and phylogenetic data, we propose the formation of a new genus (Hepatospora) to replace the previous generic classification of the Asian parasite as Endoreticulatus. The microsporidian from the hepatopancreas of E. sinensis is named Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. It is assumed that the parasite was introduced during initial invasions of this crab to Europe during the early 20th Century.

Cytological and molecular description of Hamiltosporidium tvaerminnensis gen. et sp. nov., a microsporidian parasite of Daphnia magna, and establishment of Hamiltosporidium magnivora comb. nov.

We describe the new microsporidium Hamiltosporidium tvaerminnensis gen. et sp. nov. with an emphasis on its ultrastructural characteristics and phylogenetic position as inferred from the sequence data of SSU rDNA, alpha- and beta-tubulin. This parasite was previously identified as Octosporea bayeri Jírovec, 1936 and has become a model system to study the ecology, epidemiology, evolution and genomics of microsporidia - host interactions. Here, we present evidence that shows its differences from O. bayeri. Hamiltosporidium tvaerminnensis exclusively infects the adipose tissue, the ovaries and the hypodermis of Daphnia magna and is found only in host populations located in coastal rock pool populations in Finland and Sweden. Merogonial stages of H. tvaerminnensis have isolated nuclei; merozoites are formed by binary fission or by the cleaving of a plasmodium with a small number of nuclei. A sporogonial plasmodium with isolated nuclei yields 8 sporoblasts. Elongated spores are generated by the most finger-like plasmodia. The mature spores are polymorphic in shape and size. Most spores are pyriform (4·9-5·6×2·2-2·3 μm) and have their polar filament arranged in 12-13 coils. A second, elongated spore type (6·8-12·0×1·6-2·1 μm) is rod-shaped with blunt ends and measures 6·8-12·0×1·6-2·1 μm. The envelope of the sporophorous vesicle is thin and fragile, formed at the beginning of the sporogony. Cytological and molecular comparisons with Flabelliforma magnivora, a parasite infecting the same tissues in the same host species, reveal that these two species are very closely related, yet distinct. Moreover, both cytological and molecular data indicate that these species are quite distant from F. montana, the type species of the genus Flabelliforma. We therefore propose that F. magnivora also be placed in Hamiltosporidium gen. nov.

Morphology and taxonomy of the microsporidium Liebermannia covasacrae n. sp. from the grasshopper Covasacris pallidinota (Orthoptera, Acrididae)

During a survey for grasshopper pathogens in Argentina in 2005–2006, individual Covasacris pallidinota from halophylous grasslands in Laprida, Buenos Aires province were found to be infected with a microsporidium. Infection was restricted to the salivary gland epithelial cells. The microsporidium produced ovocylindrical spores averaging 2.6 ± 0.28 × 1.4 ± 0.12 μm (range 2.2–3.4 × 1.1–1.7 μm), which resembled in size and shape the spores of Liebermannia patagonica and L. dichroplusae, two recently described species that also parasitize Argentine grasshoppers. The life cycle of the microsporidium included the formation of polynucleate, diplokaryotic, moniliform, merogonial plasmodia wrapped in flattened cisterns of the host endoplasmic reticulum (ER). Plasmodia divided to produce diplokaryotic cells. The latter underwent elongation, dissociation of diplokarya counterparts, vacuolization, dismantling of the host ER envelope, and deposition of electron-dense material outside the plasma membrane. The resultant binucleate sporogonial plasmodia divided into two uninucleate sporoblasts, which eventually transformed into spores. Uninucleate spores contained a lamellar polaroplast, embraced by an elongated polar sac, anchoring disc, 3–5 polar filament coils, and a cluster of anastomizing tubules (sporoblast trans-Golgi, posterosome) at the posterior end. Sequence similarity of the SSU rDNA of the newly discovered microsporidium (Genbank accession no. EU709818) to L. patagonica and L. dichroplusae was 99% and 97%, respectively, suggesting that the three species belong to one genus. All three species fell into one clade in SSU rDNA-based phylogenetic trees produced by neighbor joining, maximum parsimony, and maximum likelihood analyses with 100% statistical support. We assign the name Liebermannia covasacrae to this microsporidium. It can be easily differentiated from both congeners by host species, tissue tropism, type of sporogony, and several features of morphology. Comparison of the three Liebermannia spp. demonstrates that the nuclear phase (dikaryotic versus monokaryotic spores) and type of sporogony (polysporous versus disporous) may vary in closely related species.