Molecular phylogeny and new light microscopic data of Metchnikovella spiralis (Microsporidia: Metchnikovellidae), a hyperparasite of eugregarine Polyrhabdina sp. from the polychaete Pygospio elegans.

Metchnikovellids are a deep-branching group of microsporidia, parasites of gregarines inhabiting the alimentary tract of polychaetes and some other invertebrates. The diversity and phylogeny of these hyperparasites remain poorly studied. Modern descriptions and molecular data are still lacking for many species. The results of a light microscopy study and molecular data for Metchnikovella spiralis Sokolova et al., 2014, a hyperparasite of the eugregarine Polyrhabdina sp., isolated from the polychaete Pygospio elegans, were obtained. The original description of M. spiralis was based primarily on the analysis of stained preparations and transmission electron microscopy images. Here, the species description was complemented with the results of in vivo observations and phylogenetic analysis based on the SSU rRNA gene. It was shown that in this species, free sporogony precedes sac-bound sporogony, as it occurs in the life cycle of most other metchnikovellids. Spore sacs are entwined with spirally wound cords, and possess only one polar plug. Phylogenetic analyses did not group M. spiralis with M. incurvata, another metchnikovellid from the same gregarine species, but placed it as a sister branch to Amphiacantha. The paraphyletic nature of the genus Metchnikovella was discussed. The taxonomic summary for M. spiralis was emended.

Two new species of Unikaryon (Microsporidia) hyperparasitic in microphallid metacercariae (Digenea) from Florida intertidal crabs.

The genus Unikaryon (Microsporidia) holds exclusively hyperparasites of Platyhelminthes. Four species of Unikaryon are presently known from trematodes infecting mollusks and fish, and one from a cestode infecting a fish. Here we report two species of Unikaryon from microphallid trematode metacercariae parasitizing the brachyuran crabs, Panopeus herbstii and Pachygrapsus transversus, collected from intertidal habitats in Florida. The first microsporidium, which we assign here to a new species, Unikaryon panopei sp. n., was isolated from Microphallus sp. encysted in Panopeus herbstii from Tampa Bay. The specific designation for the second Unikaryon sp. (Unikaryon sp. 2), which occurred in metacercaria of Diacetabulum sp. found in P. transversus from the Florida Keys, is pending due to the lack of SSrDNA sequence data. Light and electron microscopy demonstrates that both species display characteristics of the genus Unikaryon including the arrangement of spores in sets of two, large posterior vacuole, and eccentric position of the polar filament. Spores of Unikaryon panopei sp. n., unlike those of Unikaryon sp. 2, assemble in large membrane-bound masses containing hundreds of organisms, and display a larger number of polar filament coils - 7-8, compared to 4-5 in Unikaryon sp. 2 The SSUrDNA-inferred phylogenetic analysis places Unikaryon panopei in one clade with Unikaryon legeri, the only other molecularly characterized member of the genus, with 94% of SSUrDNA similarity. These findings increase the number of species parasitizing trematodes and broaden the host range of Unikaryon spp.

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.

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.

A novel dimorphic microsporidian Ameson iseebi sp. nov. infecting muscle of the Japanese spiny lobster, Panulirus japonicus, in western Japan.

Japanese spiny lobsters (Panulirus japonicus) exhibiting white opaque abdominal muscle were found in Mie and Wakayama prefectures, in mid-Western Japan. Microscopically, two types of microsporidian spores, ovoid and rod-shaped, were observed infecting the muscle. Histologically, both types of spore were detected inside myofibers of the abdomen, appendages, and cardiac muscles and were often both observed in a single myofiber simultaneously. Transmission electron microscopy revealed that ovoid spores have villous projections on the surface, and that ovoid and rod-shaped spores have a polar filament with 12 coils and 6 to 8 coils respectively. Merogonic and sporogonic stages were observed around ovoid spores, but rarely around rod-shaped spores. The small subunit ribosomal DNA sequences obtained from both spore types were identical to each other, indicating that this microsporidian exhibits a clear spore dimorphism. Phylogenetic analysis based on the rDNA sequences indicates that this microsporidian is part of a clade consisting of the genera Ameson and Nadelspora, with the most closely related species being A. herrnkindi found in the Caribbean spiny lobster P. argus. Based on ultrastructural features, molecular phylogenetic data, host type and geographical differences among known species in these genera, the species found in whitened abdominal muscles of the Japanese spiny lobster is described as Ameson iseebi sp. nov.

Molecular and morphological characterisation of a novel microsporidian species, Tubulinosema suzukii, infecting Drosophila suzukii (Diptera: Drosophilidae).

A microsporidium showing morphological characteristics typical of a Tubulinosema species was discovered in Drosophila suzukii. All developmental stages were diplokaryotic and grew in direct contact with the host cell cytoplasm. Spores from fresh preparations were ovoid to slightly pyriform and measured 4.29 × 2.47 µm in wet mount preparations. The spore wall consisted of a 125 nm thick endospore covered by a double layered exospore of 39 nm and 18 nm. The polar filament measured 67 µm in length, was slightly anisofilar and was arranged in ten coils in one or rarely two rows. The two posterior coils were 95 nm in diameter while the anterior coils were 115 nm in diameter. Early developmental stages were surrounded by electron-dense, 35.3 nm diameter, surface ornaments scattered over the membrane. Tubular elements with diameters of approximately 75 nm were seen attaching to the periphery of meronts and sporonts. Tissues infected included fat body, midgut and muscle. A 1915 bp rDNA fragment, covering the small subunit (SSU), the internal transcribed spacer (ITS) and the 5' end of the large subunit ribosomal DNA, was amplified by PCR and sequenced. Phylogenetic analyses of the SSU rDNA fragment revealed closest relationship to Tubulinosema pampeana (Host: Bombus atratus, South America) and Tubulinosema loxostegi (Host: Loxostege sticticalis, ubiquitous), but using the complete dataset of SSU-ITS-LSU rDNA genes revealed T. hippodamiae (Host: Hippodamiae convergens) as the most closely related species. Based on the morphological and genetic features a new species, Tubulinosema suzukii sp. nov., is proposed for this microsporidium isolated from D. suzukii.

An Ultrastructural Study of the Extruded Polar Tube of Anncaliia algerae (Microsporidia).

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 200 nm, 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 5-6 nm 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.

Clarifying the Relationships between Microsporidia and Cryptomycota.

Some protists with microsporidian-like cell biological characters, including Mitosporidium, Paramicrosporidium, and Nucleophaga, have SSU rRNA gene sequences that are much less divergent than canonical Microsporidia. We analysed the phylogenetic placement and environmental diversity of microsporidian-like lineages that group near the base of the fungal radiation and show that they group in a clade with metchnikovellids and canonical microsporidians, to the exclusion of the clade including Rozella, in line with what is currently known of their morphology and cell biology. These results show that the phylogenetic scope of Microsporidia has been greatly underestimated. We propose that much of the lineage diversity previously thought to be cryptomycotan/rozellid is actually microsporidian, offering new insights into the evolution of the highly specialized parasitism of canonical Microsporidia. This insight has important implications for our understanding of opisthokont evolution and ecology, and is important for accurate interpretation of environmental diversity. Our analyses also demonstrate that many opisthosporidian (aphelid+rozellid+microsporidian) SSU V4 OTUs from Neotropical forest soils group with the short-branching Microsporidia, consistent with the abundance of their protist and arthropod hosts in soils. This novel diversity of Microsporidia provides a unique opportunity to investigate the evolutionary origins of a highly specialized clade of major animal parasites.

Microsporidian genus Berwaldia (Opisthosporidia, Microsporidia), infecting daphnids (Crustacea, Branchiopoda): Biology, structure, molecular phylogeny and description of two new species.

Structural, molecular and life cycle data are presented for two microsporidian species of the genus Berwaldia: B. singularis Larsson, 1981 (type species of the genus) and B. schaefernai Vávra and Larsson, 1994, parasites of Daphnia pulex Leydig, 1860 and Daphnia galeata Sars, 1863, respectively. Analysis of the SSU rDNA gene confirmed the species status of both species and showed that the GenBank sequence data submitted previously in GenBank for the genus Berwaldia, are from microsporidia that are not Berwaldia. Correct SSU rDNA gene sequences for B. schaefernai and B. singularis are now deposited in GenBank. The life cycle of these two species appears incomplete as the spores collected from their respective infected hosts will not infect the same host when fed per os. B. schaefernai appears as a frequent parasite of Daphnia longispina/galeata complex daphnids, influencing the behaviour of the infected host. In addition, two new species, of Berwaldia, one infecting fat body tissues of Daphnia longispina/galeata complex, and the other, infecting hypodermis and fat cells of Simocephalus vetulus (O. F. Müller, 1776) are described.

Genome analysis and polar tube firing dynamics of mosquito-infecting microsporidia.

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

Early development and tissue distribution of Pseudoloma neurophilia in the zebrafish, Danio rerio.

The early proliferative stages of the microsporidian parasite, Pseudoloma neurophilia were visualized in larval zebrafish, Danio rerio, using histological sections with a combination of an in situ hybridization probe specific to the P. neurophilia small-subunit ribosomal RNA gene, standard hematoxylin-eosin stain, and the Luna stain to visualize spores. Beginning at 5 d post fertilization, fish were exposed to P. neurophilia and examined at 12, 24, 36, 48, 72, 96, and 120 h post exposure (hpe). At 12 hpe, intact spores in the intestinal lumen and proliferative stages developing in the epithelial cells of the anterior intestine and the pharynx and within hepatocytes were observed. Proliferative stages were visualized in the pancreas and kidney at 36-48 hpe and in the spinal cord, eye, and skeletal muscle beginning at 72 hpe. The first spore stages of P. neurophilia were observed at 96 hpe in the pharyngeal epithelium, liver, spinal cord, and skeletal muscle. The parasite was only observed in the brain of larval fish at 120 hpe. The distribution of the early stages of P. neurophilia and the lack of mature spores until 96 hpe indicates that the parasite gains access to organs distant from the initial site of entry, likely by penetrating the intestinal wall with the polar tube.

Plastic parasites: extreme dimorphism creates a taxonomic conundrum in the phylum Microsporidia.

In this paper, we combine field observations of highly statistically significant co-occurrence with histopathological, ultrastructural and molecular phylogenetic analyses, to provide evidence for extreme morphological plasticity in a microsporidium parasite infecting the musculature of marine crabs. The parasite appears to alternate between lineages that culminate in production of either bizarre needle-like spores in the peripheral sarcoplasm of heart and skeletal muscle fibres (reminiscent of Nadelspora canceri infecting Cancer magister) or alternatively, Ameson-like spores with pronounced surface projections, in the skeletal muscles (as for Ameson pulvis, previously described infecting Carcinus maenas). Both lineages occur in direct contact with the cytoplasm of host muscle cells and can exist simultaneously within the same cell. Pathological data appears to reveal a remarkable shift in morphology during pathogenic remodelling of host tissues. Sequence analysis of multiple clones derived from amplification of the ssrRNA gene from infected regions of the heart and skeletal muscles appear to confirm the genetic identity of the two lineages. Furthermore, derived ssrRNA gene sequences are more similar (>99%) to N. canceri than to the coparasite Ameson michaelis infecting Callinectes sapidus (93%). Although molecular phylogenetic data support transfer of A. pulvis into the genus Nadelspora, the expansion in the generic description required to include such widely divergent characteristics is so significant as to be unfeasible within the current taxonomic framework of the phylum Microsporidia. At present, it is preferable to propose that the parasite infecting C. maenas forms a clade with other morphologically diverse but phylogenetically and ecologically similar muscle-infecting microsporidians from marine crustacean hosts. Given the strong evidence for significant plasticity in morphology amongst members of the phylum Microsporidia, novel approaches to phylogeny, based predominantly upon the informed use of molecular sequence data, are now deemed a necessity.

Phylogenetic characterization of a microsporidium (Endoreticulatus sp. Zhenjiang) isolated from the silkworm, Bombyx mori.

This study examined the morphological and molecular characteristics of the microsporidium Endoreticulatus sp. Zhenjiang, isolated from the silkworm (Bombyx mori). The fresh spores were oval, 2.9 ± 0.2 µm in length and 1.2 ± 0.2 µm in width. The complete rRNA cistron has a length of 4,432 bp (GenBank accession no. FJ772431), including the large subunit rRNA (2,460 bp), the internal transcribed spacer (187 bp), the small subunit rRNA (1,254 bp), the intergenic spacer (276 bp), and the 5S region (115 bp). The organization of the rRNA gene is 5'-LSU-ITS-SSU-IGS-5S-3', which is reverse compared to the organization of most microsporidian rRNA regions. Phylogenetic analysis based on small subunit rRNA sequences showed that this isolate belongs to the genus Endoreticulatus, and is closely related to Glugoides intestinalis. Furthermore, both had a similar reverse arrangement of the rRNA gene. Our study provides another example of a microsporidian species with a novel organization of rRNA genes, demonstrating that the reverse arrangement is exhibited not only by the microsporidian genus Nosema but may also occur in a clade that contains the genera Endoreticulatus and Glugoides.

Kneallhazia carolinensae sp. nov., a microsporidian pathogen of the thief ant, Solenopsis carolinensis.

A new species of microsporidia is described from adults of the thief ant, Solenopsis carolinensis, collected in Florida, USA. Morphological and genetic characterization of this new species showed that it is most closely related to the genus Kneallhazia and is therefore formally designated, Kneallhazia carolinensae sp. nov. Masses of ovoid, binucleate spores were localized to fat body of adult workers and measured 6.2±0.1×3.1±0.1 µm (fresh) and 6.0±0.1×3.4±0.1 µm (fixed). These spores were in direct contact with the cell cytoplasm and contained an isofilar polar filament with 12-15 coils. Blastn analysis revealed that the K. carolinensae 16S rDNA sequence exhibited 91% identity with the 16S rDNA gene of K. solenopsae. The morphological and sequence data support the conclusion that K. carolinensae is a novel microsporidian species distinct from K. solenopsae.

[A new microsporidian species Glugea mesocotti sp. n. (Microsporidia:Glugeidae) from Mesocottus haitej (Scorpaeniformes:Cottidae)].

A new microsporidian parasite of a freshwater fish Mesocottus haitej from the Amur River basin of Russia is described using light microscopy. The numerous whitish xenomas, round or oval, up to 3.0 mm large were found to be located in subcutaneous tissue of the body and mouth cavity, in the intestine and other internal organs. The formol fixed spores are elongate oval, measuring 4.8 (4.5-5.0) x 2.3 (2.2-2.5) microm in a wet smears. Posterior vacuole occupies about half of the spores. Sporophorous vesicles measuring up to 13 microm contain a great number of spores.

Microsporidia: a model for minimal parasite-host interactions.

Microsporidia are emerging fungi-like intracellular parasites of economic, veterinary and medical importance. The strategy they use to invade their host is related to the rapid extrusion of a unique and highly specialized organelle, the polar tube, which allows the injection of the infectious spore content within a target cell. This original process seems to be dependent on initial interactions between parasite and host cell components. The extreme reduction and compaction of most microsporidian genomes resulted in the loss of many metabolic pathways, which makes these parasites highly dependent on their host. Recent significant advances have been made in the understanding of mammal and insect immune responses against microsporidian infections with the involvement of both adaptive and innate immunity.

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 x 1.4+/-0.12 microm (range 2.2-3.4 x 1.1-1.7 microm), 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.

Animal cell cultures in microsporidial research: their general roles and their specific use for fish microsporidia.

The use of animal cell cultures as tools for studying the microsporidia of insects and mammals is briefly reviewed, along with an in depth review of the literature on using fish cell cultures to study the microsporidia of fish. Fish cell cultures have been used less often but have had some success. Very short-term primary cultures have been used to show how microsporidia spores can modulate the activities of phagocytes. The most successful microsporidia/fish cell culture system has been relatively long-term primary cultures of salmonid leukocytes for culturing Nucleospora salmonis. Surprisingly, this system can also support the development of Enterocytozoon bienusi, which is of mammalian origin. Some modest success has been achieved in growing Pseudoloma neurophilia on several different fish cell lines. The eel cell line, EP-1, appears to be the only published example of any fish cell line being permanently infected with microsporidia, in this case Heterosporis anguillarum. These cell culture approaches promise to be valuable in understanding and treating microsporidia infections in fish, which are increasingly of economic importance.

Morphology, molecular phylogeny, and ecology of Binucleata daphniae n. g., n. sp. (Fungi: Microsporidia), a parasite of Daphnia magna Straus, 1820 (Crustacea: Branchiopoda).

We describe a new microsporidian species Binucleata daphniae, n. g., n. sp., that infects the integument cells lining the hemocoele cavity of the carapace and the postabdomen of the cladoceran Daphnia magna Straus. Infected cells filled with spores accumulate as large clusters in the carapace cavity and heavily infected hosts are detected by their opaque appearance. Despite the parasite's presence, infected Daphnia grow and molt, but have a reduced fecundity. During the parasite's life cycle, chain-like meronts with isolated nuclei are formed, giving rise to binucleate presporonts, the most frequently observed, characteristic developmental stage. In sporogony, the nuclei of the presporont separate, divide, and eight spores enclosed in a thin-walled sporophorous vesicle are formed. Spores are 4.9 x 2.5 microm in size (fresh) and have an anisofilar polar filament with eight coils. DNA sequence analysis places B. daphniae in a clade of microsporidians that parasitize crustaceans and mosquitoes and have assumed complex life cycles. Binucleata daphniae, however, has a simple and direct life cycle and can be transferred to naïve hosts and maintained as persistent infections in populations of its host D. magna. We propose that B. daphniae has simplified its life cycle by losing its secondary host, rendering it unique in this clade.