Isolation and Identification of Nematode-Infecting Microsporidia.

Nematodes are naturally infected by the fungal-related pathogen microsporidia. These ubiquitous eukaryotic parasites are poorly understood, despite infecting most types of animals. Identifying novel species of microsporidia and studying them in an animal model can expedite our understanding of their infection biology and evolution. Nematodes present an excellent avenue for pursuing such work, as they are abundant in the environment and many species are easily culturable in the laboratory. The protocols presented here describe how to isolate bacterivorous nematodes from rotting substrates, screen them for microsporidia infection, and molecularly identify the nematode and microsporidia species. Additionally, we detail how to remove environmental contaminants and generate a spore preparation of microsporidia from infected samples. We also discuss potential pitfalls and provide suggestions on how to mitigate them. These protocols allow for the identification of novel microsporidia species, which can serve as an excellent starting point for genomic analysis, determination of host specificity, and infection characterization. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Gathering samples Support Protocol 1: Generating 10× and 40× Escherichia coli OP50 and seeding NGM plates Basic Protocol 2: Microsporidia screening, testing for Caenorhabditis elegans susceptibility, and sample freezing Basic Protocol 3: DNA extraction, PCR amplification, and sequencing to identify nematode and microsporidia species Basic Protocol 4: Removal of contaminating microbes and preparation of microsporidia spores Support Protocol 2: Bleach-synchronizing nematodes.

Metabarcoding reveals low prevalence of microsporidian infections in castor bean tick (Ixodes ricinus).

BACKGROUND: Microsporidia is a large group of eukaryotic obligate intracellular spore-forming parasites, of which 17 species can cause microsporidiosis in humans. Most human-infecting microsporidians belong to the genera Enterocytozoon and Encephalitozoon. To date, only five microsporidian species, including Encephalitozoon-like, have been found in hard ticks (Ixodidae) using microscopic methods, but no sequence data are available for them. Furthermore, no widespread screening for microsporidian-infected ticks based on DNA analysis has been carried out to date. Thus, in this study, we applied a recently developed DNA metabarcoding method for efficient microsporidian DNA identification to assess the role of ticks as potential vectors of microsporidian species causing diseases in humans. METHODS: In total, 1070 (493 juvenile and 577 adult) unfed host-seeking Ixodes ricinus ticks collected at urban parks in the city of Poznan, Poland, and 94 engorged tick females fed on dogs and cats were screened for microsporidian DNA. Microsporidians were detected by PCR amplification and sequencing of the hypervariable V5 region of 18S rRNA gene (18S profiling) using the microsporidian-specific primer set. Tick species were identified morphologically and confirmed by amplification and sequencing of the shortened fragment of cytochrome c oxidase subunit I gene (mini-COI). RESULTS: All collected ticks were unambiguously assigned to I. ricinus. Potentially zoonotic Encephalitozoon intestinalis was identified in three fed ticks (3.2%) collected from three different dogs. In eight unfed host-seeking ticks (0.8%), including three males (1.1%), two females (0.7%) and three nymphs (0.7%), the new microsporidian sequence representing a species belonging to the genus Endoreticulatus was identified. CONCLUSIONS: The lack of zoonotic microsporidians in host-seeking ticks suggests that I. ricinus is not involved in transmission of human-infecting microsporidians. Moreover, a very low occurrence of the other microsporidian species in both fed and host-seeking ticks implies that mechanisms exist to defend ticks against infection with these parasites.

Molecular phylogeny of heritable symbionts and microbiota diversity analysis in phlebotominae sand flies and Culex nigripalpus from Colombia.

BACKGROUND: Secondary symbionts of insects include a range of bacteria and fungi that perform various functional roles on their hosts, such as fitness, tolerance to heat stress, susceptibility to insecticides and effects on reproduction. These endosymbionts could have the potential to shape microbial communites and high potential to develop strategies for mosquito-borne disease control. METHODOLOGY/PRINCIPAL FINDINGS: The relative frequency and molecular phylogeny of Wolbachia, Microsporidia and Cardinium were determined of phlebotomine sand flies and mosquitoes in two regions from Colombia. Illumina Miseq using the 16S rRNA gene as a biomarker was conducted to examine the microbiota. Different percentages of natural infection by Wolbachia, Cardinium, and Microsporidia in phlebotomines and mosquitoes were detected. Phylogenetic analysis of Wolbachia shows putative new strains of Lutzomyia gomezi (wLgom), Brumptomyia hamata (wBrham), and a putative new group associated with Culex nigripalpus (Cnig) from the Andean region, located in Supergroup A and Supergroup B, respectively. The sequences of Microsporidia were obtained of Pi. pia and Cx. nigripalpus, which are located on phylogeny in the IV clade (terrestrial origin). The Cardinium of Tr. triramula and Ps. shannoni were located in group C next to Culicoides sequences while Cardinium of Mi. cayennensis formed two putative new subgroups of Cardinium in group A. In total were obtained 550 bacterial amplicon sequence variants (ASVs) and 189 taxa to the genus level. The microbiota profiles of Sand flies and mosquitoes showed mainly at the phylum level to Proteobacteria (67.6%), Firmicutes (17.9%) and Actinobacteria (7.4%). High percentages of relative abundance for Wolbachia (30%-83%) in Lu. gomezi, Ev. dubitans, Mi. micropyga, Br. hamata, and Cx. nigripalpus were found. ASVs assigned as Microsporidia were found in greater abundance in Pi. pia (23%) and Cx. nigripalpus (11%). An important finding is the detection of Rickettsia in Pi. pia (58,8%) and Bartonella sp. in Cx. nigripalpus. CONCLUSIONS/SIGNIFICANCE: We found that Wolbachia infection significantly decreased the alpha diversity and negatively impacts the number of taxa on sand flies and Culex nigripalpus. The Principal Coordinate Analysis (PCoA) is consistent, which showed statistically significant differences (PERMANOVA, F = 2.4744; R2 = 0.18363; p-value = 0.007) between the microbiota of sand flies and mosquitoes depending on its origin, host and possibly for the abundance of some endosymbionts (Wolbachia, Rickettsia).

Panopeispora mellora n. gen. n. sp. (microsporidia) infecting Say's crab (Dyspanopeus sayi) from the Atlantic shoreline of Canada.

Say's mud crab, Dyspanopeus sayi (Brachyura: Panopeidae) is a native shallow subtidal and inter-tidal inhabitant of the Atlantic coastline of North America and an invasive species in the Mediterranean and Black Seas. Little is known about the microparasites of this host and the broader Panopeidae. We describe a novel microsporidian parasite infecting the musculature of D. sayi from Malagash, Nova Scotia (Canada), at a prevalence of 7%. Histopathology and molecular diagnostics were used to describe pathology and parasite phylogenetics, respectively. Based on SSU rDNA gene sequencing we propose that the microsporidian requires establishment of a new genus (Panopeispora n. gen.) and species (Panopeispora mellora n. sp.), due to significant differences to closest known taxa (e.g. Facilispora margolisi [81% similarity] and Thelohania butleri [80% similarity]), residing in Clade V of the Microsporidia. Archived, wax-embedded histological material was re-processed for transmission electron microscopy to obtain preliminary details of its intracellular development cycle and ultrastructure within the host musculature. The discovery of this pathogen is discussed with relevance to microsporidian taxonomy and the potential for achieving ultrastructural data from archived material.

Generation of a Microsporidia Species Attribute Database and Analysis of the Extensive Ecological and Phenotypic Diversity of Microsporidia.

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

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.

MICROSPORIDIA-ASSOCIATED GRANULOMATOUS DISEASE IN TWO AUSTRALIAN COLLECTIONS OF CAPTIVE INLAND BEARDED DRAGONS (POGONA VITTICEPS).

Microsporidia are obligate, intracellular fungi. In reptiles, they are most commonly reported in squamates. We report the first detection of microsporidiosis in inland bearded dragons (Pogona vitticeps) from Australia, and for the first time, mixed infections of microsporidium and adenovirus in asymptomatic inland bearded dragons. In one collection there were five individuals, one of which was lethargic, inappetent, and had lost weight. Two large ovarian granulomas were palpated (42 × 23 mm and 26 × 19 mm) and were surgically removed. This animal died shortly after surgery. Histological evaluation of these granulomas revealed granulomatous inflammation within or adjacent to ovarian tissue, containing numerous aggregates of microorganisms consistent with microsporidia. The organisms were confirmed as Encephalitozoon pogonae by polymerase chain reaction (PCR) and sequencing. Agamid adenovirus-1 was also detected. These two infectious agents were also detected by PCR in all the other bearded dragons in this collection (n = 5), all of which were asymptomatic. A single dragon from a second collection presented for a routine wellness examination after the sudden death of another dragon in the collection. This dragon had similar intracelomic masses to the dragon from the first collection. These were removed surgically, but the dragon died 5 wk later following 3 wk of treatment with 25 mg/kg fenbendazole PO q7 days. Necropsy samples were collected and the microsporidian Encephalitozoon pogonae was detected in oral-cloacal swabs, blood, and multiple tissues by PCR and sequencing. Agamid adenovirus-1 was not detected in this dragon.

The largest meta-analysis on the global prevalence of microsporidia in mammals, avian and water provides insights into the epidemic features of these ubiquitous pathogens.

BACKGROUND: Microsporidia are obligate intracellular parasites that can infect nearly all invertebrates and vertebrates, posing a threat to public health and causing large economic losses to animal industries such as those of honeybees, silkworms and shrimp. However, the global epidemiology of these pathogens is far from illuminated. METHODS: Publications on microsporidian infections were obtained from PubMed, Science Direct and Web of Science and filtered according to the Newcastle-Ottawa Quality Assessment Scale. Infection data about pathogens, hosts, geography and sampling dates were manually retrieved from the publications and screened for high quality. Prevalence rates and risk factors for different pathogens and hosts were analyzed by conducting a meta-analysis. The geographic distribution and seasonal prevalence of microsporidian infections were drawn and summarized according to sampling locations and date, respectively. RESULTS: Altogether, 287 out of 4129 publications up to 31 January 2020 were obtained and met the requirements, from which 385 epidemiological data records were retrieved and effective. The overall prevalence rates in humans, pigs, dogs, cats, cattle, sheep, nonhuman primates and fowl were 10.2% [2429/30,354; 95% confidence interval (CI) 9.2-11.2%], 39.3% (2709/5105; 95% CI 28.5-50.1%), 8.8% (228/2890; 95% CI 5.1-10.1%), 8.1% (112/1226; 95% CI 5.5-10.8%), 16.6% (2216/12,175; 95% CI 13.5-19.8%), 24.9% (1142/5967; 95% CI 18.6-31.1%), 18.5% (1388/7009; 95% CI 13.1-23.8%) and 7.8% (725/9243; 95% CI 6.4-9.2%), respectively. The higher prevalence in pigs suggests that routine detection of microsporidia in animals should be given more attention, considering their potential roles in zoonotic disease. The highest rate was detected in water, 58.5% (869/1351; 95% CI 41.6-75.5%), indicating that water is an important source of infections. Univariate regression analysis showed that CD4+ T cell counts and the living environment are significant risk factors for humans and nonhuman primates, respectively. Geographically, microsporidia have been widely found in 92 countries, among which Northern Europe and South Africa have the highest prevalence. In terms of seasonality, the most prevalent taxa, Enterocytozoon bieneusi and Encephalitozoon, display different prevalence trends, but no significant difference between seasons was observed. In addition to having a high prevalence, microsporidia are extremely divergent because 728 genotypes have been identified in 7 species. Although less investigated, microsporidia coinfections are more common with human immunodeficiency virus and Cryptosporidium than with other pathogens. CONCLUSIONS: This study provides the largest-scale meta-analysis to date on microsporidia prevalence in mammals, birds and water worldwide. The results suggest that microsporidia are highly divergent, widespread and prevalent in some animals and water and should be further investigated to better understand their epidemic features.

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.

Inferring the Deep Past from Molecular Data.

There is an expectation that analyses of molecular sequences might be able to distinguish between alternative hypotheses for ancient relationships, but the phylogenetic methods used and types of data analyzed are of critical importance in any attempt to recover historical signal. Here, we discuss some common issues that can influence the topology of trees obtained when using overly simple models to analyze molecular data that often display complicated patterns of sequence heterogeneity. To illustrate our discussion, we have used three examples of inferred relationships which have changed radically as models and methods of analysis have improved. In two of these examples, the sister-group relationship between thermophilic Thermus and mesophilic Deinococcus, and the position of long-branch Microsporidia among eukaryotes, we show that recovering what is now generally considered to be the correct tree is critically dependent on the fit between model and data. In the third example, the position of eukaryotes in the tree of life, the hypothesis that is currently supported by the best available methods is fundamentally different from the classical view of relationships between major cellular domains. Since heterogeneity appears to be pervasive and varied among all molecular sequence data, and even the best available models can still struggle to deal with some problems, the issues we discuss are generally relevant to phylogenetic analyses. It remains essential to maintain a critical attitude to all trees as hypotheses of relationship that may change with more data and better methods.

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.

Evolutionary relationships of Metchnikovella dogieli Paskerova et al., 2016 (Microsporidia: Metchnikovellidae) revealed by multigene phylogenetic analysis.

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

Identification and characterization of three microsporidian genera concurrently infecting a silkworm, Bombyx mori, in Brazil.

Microsporidia are important entomopathogens known for infecting insects such as the silkworm (Bombyx mori) thus impairing global silk production. This study aimed to identify and characterize the microsporidia isolated from a diseased larva of silkworm, collected from a sericulture farm in southern Brazil. Identification was performed by phylogenetic analysis of the nucleotide sequences of the SSU rRNA genes. Characterization was performed by analyzing spore sizes, tissue tropism, internal and external symptoms, and pathogenicity against B. mori. Microsporidia belonging to three different genera were identified, namely, Endoreticulatus, Nosema and Tubulinosema. After inoculation of the mixed spores of the microsporidian isolates into B. mori larvae, a high prevalence of Tubulinosema spp. was observed. This isolate showed high prevalence on the silk glands and a late mortality, initially of around 10% until the 20th day post-inoculation but reaching 91.5% upon pupation. Therefore, we demonstrated that Tubulinosema spp. causes chronic infection with slow pathogenicity. We identified for the first time three different microsporidians concurrently infecting B. mori in Brazil. Tubulinosema is of particular interest because of its potential threat to silk production; it affects the formation of silk glands in B. mori while not presenting distinguishable external symptoms or causing the immediate death of these insects. Further studies focusing on this species, mainly regarding its life cycle within the host and the sublethal effects of surviving individuals, demonstrate the importance of describing it as a new species and improving the characterization of the disease in order to prevent its spread.

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.

Complete genome of a unicellular parasite (Antonospora locustae) and transcriptional interactions with its host locust.

Microsporidia are a large group of unicellular parasites that infect insects and mammals. The simpler life cycle of microsporidia in insects provides a model system for understanding their evolution and molecular interactions with their hosts. However, no complete genome is available for insect-parasitic microsporidian species. The complete genome of Antonospora locustae, a microsporidian parasite that obligately infects insects, is reported here. The genome size of A. locustae is 3 170 203 nucleotides, composed of 17 chromosomes onto which a total of 1857 annotated genes have been mapped and detailed. A unique feature of the A. locustae genome is the presence of an ultra-low GC region of approximately 25 kb on 16 of the 17 chromosomes, in which the average GC content is only 20 %. Transcription profiling indicated that the ultra-low GC region of the parasite could be associated with differential regulation of host defences in the fat body to promote the parasite's survival and propagation. Phylogenetic gene analysis showed that A. locustae, and the microsporidian family in general, is likely at an evolutionarily transitional position between prokaryotes and eukaryotes, and that it evolved independently. Transcriptomic analysis showed that A. locustae can systematically inhibit the locust phenoloxidase PPO, TCA and glyoxylate cycles, and PPAR pathways to escape melanization, and can activate host energy transfer pathways to support its reproduction in the fat body, which is an insect energy-producing organ. Our study provides a platform and model for studies of the molecular mechanisms of microsporidium-host interactions in an energy-producing organ and for understanding the evolution of microsporidia.

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.

Molecular detection and genotyping of microsporidia species in chickens in Turkey.

Microsporidia are obligate intracellular pathogens that infect various hosts including invertebrates and vertebrates. Despite the importance, knowledge on the prevalence and molecular characteristics of microsporidia in chickens is limited, and no data are available for Turkey. A total of 300 fecal samples from chickens in the Central Anatolia Region of Turkey were analyzed by using a nested polymerase chain reaction assay targeting the rRNA internal transcribed spacer (ITS) region for the common microsporidia species. Corresponding PCR amplicons from the positive samples were sequenced for genotyping. Enterocytozoon bieneusi was identified in 22 (7.3 %) samples, whereas Encephalitozoon spp. was not detected. The prevalence of E. bieneusi was 63.6 % in Kayseri and 36.4 % in Nevsehir provinces, and 8.8 % in soft fecal samples and 9.7 % in diarrhoeic samples. No infections were found in Kirsehir Province. Significant differences were found for the distribution of E. bieneusi among provinces and fecal conditions. Infections were found only in free-range chickens. As a result of ITS region sequencing, two genotypes were characterized. The novel genotype ERUNT1 (n = 21), belonging to zoonotic group 1, was the most common genotype throughout the study area. The other known genotype, ERUSS1 (n = 1), had a restricted distribution and was previously detected in cattle and sheep in the same region. Our study provides the first data on microsporidia species from chickens in Turkey. None of these genotypes have been reported in humans; thus, the risk potential for public health is limited but needs further investigation.

Zoonotic Transmission of Cryptosporidium and Microsporidia in Individuals of the Kurdistan Province, West of Iran.

Cryptosporidium species and microsporidia, which can cause zoonotic intestinal infections in humans, have become an emerging public health concern. It seems that the identification and genotyping of these parasites are necessary for the prevention, control, and establishment of appropriate treatment. This study aimed to evaluate the distribution and zoonotic transmission routes of Cryptosporidium species and microsporidia to humans referred to medical laboratories of Kurdistan Province, Iran. A total of 1,383 stool samples were collected and investigated. Cryptosporidium spp. and microsporidia were detected using microscopic methods (i.e., formol-ether concentration, Ziehl-Neelsen staining, and modified trichrome staining methods). DNA was extracted from positive samples, and specific fragments of the Cryptosporidium GP60 gene and microsporidia SSU rRNA gene were amplified. Furthermore, positive samples were sequenced for genotype identification and bioinformatics analysis. Based on the microscopic analysis of 1,383 stool samples, 5 (0.36%) and 6 (0.43%) samples were considered positive for Cryptosporidium oocysts and microsporidia spores, respectively. Molecular analysis of positive samples identified the isolates as Cryptosporidium parvum and Enterocytozoon bieneusi. According to comparative phylogenetics, cryptosporidiosis and microsporidiosis may occur via zoonotic transmission in this region. Therefore, proper control and health education are strongly recommended to prevent zoonotic diseases.