Establishment of a TaqMan probe-based qPCR assay for detecting microsporidia Enterospora epinepheli in grouper.

Enterospora epinepheli is an intranuclear microsporidian parasite causing serious emaciative disease in hatchery-bred juvenile groupers (Epinephelus spp.). Rapid and sensitive detection is urgently needed as its chronic infection tends to cause emaciation as well as white faeces syndrome and results in fry mortality. This study established a TaqMan probe-based real-time quantitative PCR assays targeting the small subunit rRNA (SSU) gene of E. epinepheli. The relationship between the standard curve of cycle threshold (Ct) and the logarithmic starting quantity (SQ) was determined as Ct = -3.177 lg (SQ) + 38.397. The correlation coefficient (R2 ) was 0.999, and the amplification efficiency was 106.4%. The detection limit of the TaqMan probe-based qPCR assay was 1.0 × 101 copies/µL and that is 100 times sensitive than the traditional PCR method. There is no cross-reaction with other aquatic microsporidia such as Ecytonucleospora hepatopenaei, Nucleospora hippocampi, Potaspora sp., Ameson portunus. The intra-assay and inter-assay showed great repeatability and reproducibility. In addition, the test of clinical samples showed that this assay effectively detected E. epinepheli in the grouper's intestine tissue. The established TaqMan qPCR assays will be a valuable diagnostic tool for the epidemiological investigation as well as prevention and control of E. epinepheli.

Ecytonucleospora hepatopenaei n. gen. et comb. (Microsporidia: Enterocytozoonidae): A redescription of the Enterocytozoon hepatopenaei (Tourtip et al., 2009), a microsporidian infecting the widely cultivated shrimp Penaeus vannamei.

The microsporidian Enterocytozoon hepatopenaei from Penaeus vannamei (EHPPv) was redescribed on the basis of spore morphology, life cycle, pathology, and molecular character. Compared with the Enterocytozoon hepatopenaei isolated from Penaeus monodon (EHPPm), described by Tourtip et al. in 2009, new features were found in EHPPv. Electron microscopy demonstrated that EHPPv was closely associated with the nucleus of host cell. The merogony and sporogony phages were in direct contact with the cytoplasm of host cells, whereas some of the sporoblasts and the spores were surrounded by the interfacial envelope. Mature spores of EHPPv were oval and monokaryotic, measuring 1.65 ± 0.15 µm × 0.92 ± 0.05 µm. Spores possessed many polyribosomes around a bipartite polaroplast and the polar filament with 4-5 coils in two rows. Phylogenetic analyses showed all Enterocytozoon hepatopenaei isolates shared a common ancestor. Based on the morphological and molecular analyses, we propose the establishment of a new genus Ecytonucleospora and transferring Enterocytozoon hepatopenaei to the genus Ecytonucleospora, retaining the specific epithet hepatopenaei that Tourtip et al. proposed in recognition of their first research, as the new combination Ecytonucleospora hepatopenaei n. comb. Furthermore, it was suggested Enterospora nucleophila, Enterocytozoon sp. isolate RA19015_21, and Enterocytozoon schreckii be assigned into this new genus.

Morphological and molecular characterization of a new freshwater microsporidium, Jirovecia sinensis sp. n. (Microsporidia) infecting the coelomocytes of Branchiura sowerbyi (Oligochaeta: Naididae) in China.

We report a new microsporidium Jirovecia sinensis sp. n. from a freshwater oligochaete, Branchiura sowerbyi collected in Hongze city, Jiangsu province, East China. Numerous whitish hypertrophied coelomocytes of 0.33-0.59 mm in diameter indicated infection. Transmission electron microscopy observations revealed that all developmental stages were diplokaryotic. The earliest life stages observed were meronts that were in direct contact with host cytoplasm, accumulated peripherally in the hypertrophied coelomocytes and connected with host cytoplasm through many pinocytotic canals. Mature spores are rod-shaped with a blunt end, measuring 17.0 ± 0.1 (14.9-18.5) µm long and 2.0 ± 0.2 (1.7-2.2) µm wide. The most conspicuous character of the novel microsporidian parasite is the tail-like posterior prolongations, with a length of 29.6-40.8 µm. Mature spores have a manubrium with a diameter of 447-485 nm which consist of six density-discontinuous concentric circles. Spores possess a collar-shaped anchoring disk and a bipartite polarplast with an anterior lamellar region and a posterior tubular section. SSU rDNA-based phylogenetic analysis indicated with high support values that the new species clustered with two Bacillidium species (B. vesiculoformis and Bacillidium sp.) infecting the freshwater oligochaetes and Janacekia debaisieuxi infecting the insect Simulium maculatum. Based on the ultrastructural features and molecular characteristics, a new species in the genus Jirovecia, Jirovecia sinensis sp. n., is designated.

An integrated metabolic consequence of Hepatospora eriocheir infection in the Chinese mitten crab Eriocheir sinensis.

Despite the economic and evolutionary importance of aquatic host-infecting microsporidian species, at present, limited information has been provided about the microsporidia-host interactions. This study focused on Hepatospora eriocheir, an emerging microsporidian pathogen for the Chinese mitten crab Eriocheir sinensis. Hypertrophy of hepatopancreas cells was a common feature of H. eriocheir infection. More importantly, mitochondria of the hepatopancreas were drawn around the H. eriocheir, most likely to aid the uptake of ATP directly from the host. To better understand the crab anti-microsporidian response, de novo transcriptome sequencing of the hepatopancreas tissue was furtherly proceeded. A total of 47.84 M and 57.21 M clean reads were generated from the hepatopancreas of H. eriocheir infected and control groups respectively. Based on homology searches, functional annotation with 6 databases (Nr, Swiss-Prot, KEGG, KOGs, Pfam and GO) for 88,168 unigenes was performed. 2619 genes were identified as differently up-regulated and 2541 genes as differently down-regulated. Prominent functional categories enriched with differentially expressed genes (DEGs) were "ATP binding", "mitochondrion and extracellular region", "oxygen transporter activity", "oxidoreductase activity", "alanine, aspartate and glutamate metabolism", "carbohydrate metabolic process", "starch and sucrose metabolism" and "fatty acid biosynthesis". These results confirmed a parasite external energy supply and an integrated metabolic stress. In addition, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were also identified from the gene library. Taken together, these findings allow us to better understand the underlying mechanisms regulating interactions between H. eriocheir and the crab E. sinensis.

Development of In situ hybridization and real-time PCR assays for the detection of Hepatospora eriocheir, a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis.

A microsporidian parasite, Hepatospora eriocheir, is an emerging pathogen for the Chinese mitten crab Eriocheir sinensis. Currently, there is scant information about the way it transmits infection in the crustacean of commercial importance, including its pathogenesis, propagation and infection route in vivo. In this study, chromogenic in situ hybridization (ISH) and quantitative real-time PCR (qPCR) assays were developed to address this pressing need, and we provided an advance in the detection methods available. Pathogens can be seen in situ with associated lesions using ISH. Positive hybridization signals were noted inside the epithelial cells of the hepatopancreas, and putative free parasite spores were observed within the tubule lumen, which were associated with lesions detected by electron microscopy and haematoxylin and eosin (H&E) analysis. qPCR allows the determination of parasite loads in infected tissues, which is important for understanding disease progression and transmission. The hepatopancreas displayed the biggest statistical copy numbers among different tissues of infected crabs, confirming a tissue-specific pathogen infection characteristic. The qPCR assay also proved to be suitable for the diagnosis of asymptomatic carrier crabs. Combination of the two methods could facilitate the study of H. eriocheir infection mechanism in E. sinensis, enhance the early diagnosis of the pathogen and improve the management of microsporidian diseases in commercial crustaceans.

Microsporidians xenomas of anglerfish from NE Atlantic waters.

The presence of emergent visible parasites at commercial valuable fish species is increasingly causing problems at fisheries and seafood industries. Microsporidians have been previously reported to appear forming apparent xenomas complexes in anglerfish species, but no effort has been carried out to simultaneously integrate epidemiological data, phenotypic, genotypic and fine structural characterizations in the same parasite sample. In this work, specimens of Lophius budegassa and Lophius piscatorius from NE Atlantic waters were sampled and examined to provide information about specific site of infection and demographic data of two groups of different sizes of xenomas present at both fish species. Histological descriptions and scanning and transmission electron microscopy were carried out on fresh spores of Lophius budegassa for ultrastructural studies. In both types of xenomas, it was observed simultaneously the microsporidian genus Spraguea in the form of two different types of spores. Molecular analyses of both xenomas from the two fish species, based on the small subunit ribosomal DNA gene, were also performed to genetically support the morphological diagnostic provided.

The genome of Spraguea lophii and the basis of host-microsporidian interactions.

Microsporidia are obligate intracellular parasites with the smallest known eukaryotic genomes. Although they are increasingly recognized as economically and medically important parasites, the molecular basis of microsporidian pathogenicity is almost completely unknown and no genetic manipulation system is currently available. The fish-infecting microsporidian Spraguea lophii shows one of the most striking host cell manipulations known for these parasites, converting host nervous tissue into swollen spore factories known as xenomas. In order to investigate the basis of these interactions between microsporidian and host, we sequenced and analyzed the S. lophii genome. Although, like other microsporidia, S. lophii has lost many of the protein families typical of model eukaryotes, we identified a number of gene family expansions including a family of leucine-rich repeat proteins that may represent pathogenicity factors. Building on our comparative genomic analyses, we exploited the large numbers of spores that can be obtained from xenomas to identify potential effector proteins experimentally. We used complex-mix proteomics to identify proteins released by the parasite upon germination, resulting in the first experimental isolation of putative secreted effector proteins in a microsporidian. Many of these proteins are not related to characterized pathogenicity factors or indeed any other sequences from outside the Microsporidia. However, two of the secreted proteins are members of a family of RICIN B-lectin-like proteins broadly conserved across the phylum. These proteins form syntenic clusters arising from tandem duplications in several microsporidian genomes and may represent a novel family of conserved effector proteins. These computational and experimental analyses establish S. lophii as an attractive model system for understanding the evolution of host-parasite interactions in microsporidia and suggest an important role for lineage-specific innovations and fast evolving proteins in the evolution of the parasitic microsporidian lifecycle.

First case of hepatopancreatic necrosis disease in pond-reared Chinese mitten crab, Eriocheir sinensis, associated with microsporidian.

An epidemic of hepatopancreatic necrosis disease (HPND) with a high mortality rate (40%-50%) recently occurred in the cultured Chinese mitten crab, Eriocheir sinensis, which is a very important economic crustacean species in China. Histology revealed infection by a microsporidian parasite within the cytoplasm of the epithelial cells of the hepatopancreas. Numerous discrete inclusions in the infected cells and presumably free parasite spores were also observed. By negative staining using electron microscopy, a typical morphology of spores was observed with a protuberant front of the anchoring disc. Infection was confined to the epithelial cells of the hepatopancreas, with no other organ implicated. By sequencing the PCR products using specific primers based on conserved regions of microsporidian small subunit (18S) ribosomal DNA, it was revealed that the parasite from HPND ponds had 99% sequence identity to that of Hepatospora eriocheir. Phylogentic analysis also placed the microsporidian in the same lineage as H. eriocheir. This study reported the first case of widespread infections of H. eriocheir associated with HPND found in the pond-reared Chinese mitten crab, E. sinensis. The description of microsporidian in this important commercial host is fundamental for future consideration of factors affecting stock health and sustainability.

Co-infection of Nucleospora cyclopteri (Microsporidia) and Kudoa islandica (Myxozoa) in farmed lumpfish, Cyclopterus lumpus L., in Norway: a case report.

This study describes a co-infection of Kudoa islandica (Myxozoa) and Nucleospora cyclopteri (Microsporida) in farmed lumpfish, Cyclopterus lumpus L., in Norway. Several other parasites (Cryptocotyle sp., protozoan ciliates and Gyrodactylus sp.) were also found in gills. In June 2013, the mortality in a farmed lumpfish population increased to 65%. Lumpfish showed erratic swimming behaviour and loss of weight. At necropsy, nodules in the kidney were the only visible lesions. Histologically, all fish showed severe changes with gill inflammation and necrosis in the spleen, kidney and liver. Haemorrhages and necrosis were observed in some hearts. Intracellular microsporidians associated with the lesions were detected in most organs using histological examination and Calcofluor White. Kudoa spores were diagnosed in the skeletal muscle, but no inflammatory response was associated with the presence of the plasmodia. Comparison of 18S ribosomal DNA sequences showed 100% similarity to Kudoa islandica and Nucleospora cyclopteri. Kudoa islandica and N. cyclopteri have previously been described associated with lesions in wild lumpfish in Iceland. In the present case, N. cyclopteri is believed to be the main cause of systemic pathology. This is the first description of K. islandica and N. cyclopteri causing pathology in farmed lumpfish in Norway.

Anncaliia algerae microsporidial myositis.

The insect microsporidian Anncaliia algerae was first described in 2004 as a cause of fatal myositis in an immunosuppressed person from Pennsylvania, USA. Two cases were subsequently reported, and we detail 2 additional cases, including the only nonfatal case. We reviewed all 5 case histories with respect to clinical characteristics, diagnosis, and management and summarized organism life cycle and epidemiology. Before infection, all case-patients were using immunosuppressive medications for rheumatoid arthritis or solid-organ transplantation. Four of the 5 case-patients were from Australia. All diagnoses were confirmed by skeletal muscle biopsy; however, peripheral nerves and other tissues may be infected. The surviving patient received albendazole and had a reduction of immunosuppressive medications and measures to prevent complications. Although insects are the natural hosts for A. algerae, human contact with water contaminated by spores may be a mode of transmission. A. algerae has emerged as a cause of myositis, particularly in coastal Australia.

Pathogenesis of Experimental Infection of Nile Tilapia (Oreochromis niloticus) with Nucleospora Braziliensis Pathology and Proteomic of Microsporidia.

The recent discovery of disease caused by Nucleospora braziliensis in Nile tilapia (Oreochromis niloticus) is important as it has highlighted the high prevalence of infection and associated mortality in cultured fish. Thus, this study conducted an experimental infection of this microsporidium to evaluate pathological alterations and conduct proteomic analysis. For pathological observation, samples of brain, eyes, gall bladder, gut, heart, kidney, liver, muscle, skin, spleen, and stomach tissue, were collected, and liquid chromatography-mass spectrometry (LC-MS/MS) was performed for proteomic analysis. The most prevalent lesions were brownish color of the liver, gill filament fusion, gut ischemia, hemorrhage of the lips and fins, hepatomegaly, spleen atrophy, splenomegaly, and stomach congestion. The most common microscopic lesions were degeneration, hemorrhage, and inflammation in the brain, gills, gut, kidney, liver, muscle, spleen, and stomach. The digested peptides were identified by LC-MS/MS and the intersection of each group showed that in the spleen there were 121 exclusive proteins in the infected sample and 252 in the control, while in the kidney, 129 proteins were identified in the infected specimen compared to 83 in the control. In conclusion, this study demonstrates the proteome profile of O. niloticus kidney and spleen tissue in response to infection with N. braziliensis.

Redefining the genus Spraguea based on ultrastructural and phylogenetic data from Spraguea gastrophysus n. sp. (phylum Microsporidia), a parasite found in Lophius gastrophysus (Teleostei) from Brazil.

The ultrastructure of the fish-infecting microsporidium Spraguea gastrophysus found in the dorsal ganglia and kidney of the anglerfish, Lophius gastrophysus (family Lophiidae) collected on the Brazilian Atlantic coast is described. Each whitish xenoma (up to 3.1 × 1.8 mm) contains several groups of parasites. The host cells are hypertrophied and contain various parasite life stages including mature spores and several developmental stages with unpaired nuclei. Monomorphic spores are ellipsoidal, lightly curved and measure about 3.35 × 1.71 µm. The spore contains a gradually tapering isofilar polar filament with five to six coils arranged in a single row. The nucleus occupies a central zone of the sporoplasm where also several polyribosomes are presented. The posterior vacuole contains a voluminous spherical and granular posterosome measuring up to ~0.65 µm in diameter. The partial small subunit, intergenic spacer and partial large subunit rRNA gene were sequenced and the phylogenetic analysis places the microsporidian described here in the clade that includes all sequences of the Spraguea genus. The ultrastructural morphology of the xenoma and the spores of this microsporidian parasite, as well as the molecular and phylogenetic analysis, suggest the description of a new species. A redefining of the genus Spraguea is also done.

The Spraguea Parasitism in Anglerfishes of the Genus Lophius.

PURPOSE: To follow the development of the microsporidian Spraguea americanus within the nervous tissue of Lophius. An attempt to determine when and how the infection begins. METHODS: Acquiring different age groups of Lophius and recovering the infected sites, particularly the supramedullary neuron fibers and preparing them for microscopy. RESULTS: The youngest juvenile Lophius recovered were 140 mm long with established infections. These infections consisted of meronts and sporoblasts but no spores. The evidence indicates these infections began a month or so earlier. CONCLUSIONS: Early stages of S. americanus development occur only in juvenile Lophius and not present in older fish. The prediction is infections of all Spraguea species begin early in the life of benthic juvenile Lophius. The high incidence of infection among these fish is an indicator that the location where the infection begins is likely rich in infective spores.

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.

Spraguea (Microsporida: Spraguidae) infections in the nervous system of the Japanese anglerfish, Lophius litulon (Jordan), with comments on transmission routes and host pathology.

Anglerfish from the genus Lophius are a globally important commercial fishery. The microsporidian Spraguea infects the nervous system of these fish resulting in the formation of large, visible parasitic xenomas. Lophius litulon from Japan were investigated to evaluate the intensity and distribution of Spraguea xenomas throughout the nervous system and to assess pathogenicity to the host and possible transmission routes of the parasite. Spraguea infections in L. litulon had a high prevalence; all fish over 403 mm in standard length being infected, with larger fish usually more heavily infected than smaller fish. Seventy percent of all fish examined had some gross visible sign of infection. The initial site of development is the supramedullary cells on the dorsal surface of the medulla oblongata, where all infected fish have parasitic xenomas. As the disease progresses, a number of secondary sites typically become infected such as the spinal, trigeminal and vagus nerves. Fish with infection in the vagus nerve bundles often have simultaneous sites of infection, in particular the spinal nerves and along the ventral nerve towards the urinary bladder. Advanced vagus nerve infections sometimes form xenomas adjacent to kidney tissue. Spraguea DNA was amplified from the contents of the urinary bladders of two fish, suggesting that microsporidian spores may be excreted in the urine. We conclude that supramedullary cells on the hindbrain are the primary site of infection, which is probably initiated at the cutaneous mucous glands where supramedullary cells are known to extend their peripheral axons. The prevalence of Spraguea infections in L. litulon was very high, and infections often extremely heavy; however, no associated pathogenicity was observed, and heavily infected fish were otherwise normal.

Paranucleospora theridion n. gen., n. sp. (Microsporidia, Enterocytozoonidae) with a Life Cycle in the Salmon Louse (Lepeophtheirus salmonis, Copepoda) and Atlantic Salmon (Salmo salar).

Paranucleospora theridion n. gen, n. sp., infecting both Atlantic salmon (Salmo salar) and its copepod parasite Lepeophtheirus salmonis is described. The microsporidian exhibits nuclei in diplokaryotic arrangement during all known life-cycle stages in salmon, but only in the merogonal stages and early sporogonal stage in salmon lice. All developmental stages of P. theridion are in direct contact with the host cell cytoplasm or nucleoplasm. In salmon, two developmental cycles were observed, producing spores in the cytoplasm of phagocytes or epidermal cells (Cycle-I) and in the nuclei of epidermal cells (Cycle-II), respectively. Cycle-I spores are small and thin walled with a short polar tube, and are believed to be autoinfective. The larger oval intranuclear Cycle-II spores have a thick endospore and a longer polar tube, and are probably responsible for transmission from salmon to L. salmonis. Parasite development in the salmon louse occurs in several different cell types that may be extremely hypertrophied due to P. theridion proliferation. Diplokaryotic merogony precedes monokaryotic sporogony. The rounded spores produced are comparable to the intranuclear spores in the salmon in most aspects, and likely transmit the infection to salmon. Phylogenetic analysis of P. theridion partial rDNA sequences place the parasite in a position between Nucleospora salmonis and Enterocytozoon bieneusi. Based on characteristics of the morphology, unique development involving a vertebrate fish as well as a crustacean ectoparasite host, and the results of the phylogenetic analyses it is suggested that P. theridion should be given status as a new species in a new genus.

[Analysis of expression of vesicular transport genes in avesicular cells of the microsporidium Paranosema (Antonospora) locustae].

Long adaptation of microsporidia, a large group fungi-related protozoa, to intracellular lifestyle has resulted in a drastic minimization of parasite cell. Ultrastructural analysis has shown that the Golgi complex of the microsporidia Paranosema (Antonospora) grylli and P. locustae appears as branching or varicose networks of thin tubules. These tubular networks are connected to endoplasmic reticulum, plasma membrane and forming polar tube but have no vesicles. Vesicles were not found even if ultra-fast cryofixation and membrane fusion/uncoating inhibition were used. However, a limited number of genes involved in vesicular transport were found in microsporidia genomes. In this study we used RT-PCR to analyze the content of mRNA transcripts encoding beta and beta' subunits COPI coatomer complex, Sec13 and Sec31 subunits COPII, SNARE-proteins synaptobrevin and syntaxin-like member of SFT family in P. locustae intracellular stages. The level of expression of studied genes was comparable with that of gene encoding alternative oxidase, enzyme envolved in microsporidia core metabolism. Moreover, polyclonal antibodies raised against recombinant Sec13 subunit COPII, expressed in B Escherichia coli, has shown accumulation of the protein is spores and stages of intracellular development as well as its association with membranes. The presence of components of vesicular transport machinery in avesicular microsporidia cells requires their functional analysis.

Genome sequence surveys of Brachiola algerae and Edhazardia aedis reveal microsporidia with low gene densities.

BACKGROUND: Microsporidia are well known models of extreme nuclear genome reduction and compaction. The smallest microsporidian genomes have received the most attention, but genomes of different species range in size from 2.3 Mb to 19.5 Mb and the nature of the larger genomes remains unknown. RESULTS: Here we have undertaken genome sequence surveys of two diverse microsporidia, Brachiola algerae and Edhazardia aedis. In both species we find very large intergenic regions, many transposable elements, and a low gene-density, all in contrast to the small, model microsporidian genomes. We also find no recognizable genes that are not also found in other surveyed or sequenced microsporidian genomes. CONCLUSION: Our results demonstrate that microsporidian genome architecture varies greatly between microsporidia. Much of the genome size difference could be accounted for by non-coding material, such as intergenic spaces and retrotransposons, and this suggests that the forces dictating genome size may vary across the phylum.

Alpha- and beta-tubulin phylogenies support a close relationship between the microsporidia Brachiola algerae and Antonospora locustae.

Microsporidia are a large and diverse group of intracellular parasites related to fungi. Much of our understanding of the relationships between microsporidia comes from phylogenies based on a single gene, the small subunit (SSU) rRNA, because only this gene has been sampled from diverse microsporidia. However, SSUrRNA trees are limited in their ability to resolve basal branches and some microsporidian affiliations are inconsistent between different analyses. Protein phylogenies have provided insight into relationships within specific groups of microsporidia, but have rarely been applied to the group as a whole. We have sequenced alpha- and beta-tubulins from microsporidia from three different subgroups, including representatives from what have previously been inferred to be the basal branches, allowing the broadest sampled protein-based phylogenetic analysis to date. Although some relationships remain unresolved, many nodes uniting subgroups are strongly supported and consistent in both individual trees as well as a concatenate of both tubulins. One such relationship that was previously unclear is between Brachiola algerae and Antonospora locustae, and their close association with Encephalitozoon and Nosema. Also, an uncultivated microsporidian that infects cyclopoid copepods is shown to be related to Edhazardia aedis.