Mortality events associated with microsporidian, Spraguea sp., in White Trevally culture in Japan.

In 2021, White Trevally or Striped Jack cultured in the western part of Japan exhibited mild, but chronic mortalities from late September through early October. The cumulative mortality rate was approximately 0.02% per a net pen containing approximately 50,000 fish. Although the cumulative mortality rate was not high, most of the fish in net pens showed characteristic gross signs and an abnormal swimming behaviour. The body of diseased fish became pale and the yellow lines on the lateral sides of fish body became darken. In addition, silver lines along the dorsal fin became apparent. Loss of schooling behaviour was noted during the mortality event. In addition, affected fish became lethargic and failed to swim against current, or frequently stopped swimming and sank to the bottom of net pens after feeding. The goal of this study was to identify the cause of the mortality event. To achieve the goal, we used histopathology and metatranscriptome analysis. Histopathological examination revealed that xenoma of microsporidian were frequently observed in the nerve axon in the brain and spinal cord. Spores observed in the sections were stained with a fluorescent dye, Uvitex 2B, indicating those spores are microsporidian. The data from metatranscriptome analysis indicated that the microsporidian is Spraguea sp. The microsporidian was frequently detected from diseased fish with similar symptoms collected in the same region, suggesting that the microsporidian was highly associated with abnormal swimming behaviour of fish.

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.

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.

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.

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.

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.

In vitro propagation of the microsporidian pathogen Brachiola algerae and studies of its chromosome and ribosomal DNA organization in the context of the complete genome sequencing project.

Brachiola algerae has a broad host spectrum from human to mosquitoes. The successful infection of two mosquito cell lines (Mos55: embryonic cells and Sua 4.0: hemocyte-like cells) and a human cell line (HFF) highlights the efficient adaptive capacity of this microsporidian pathogen. The molecular karyotype of this microsporidian species was determined in the context of the B. algerae genome sequencing project, showing that its haploid genome consists of 30 chromosomal-sized DNAs ranging from 160 to 2240 kbp giving an estimated genome size of 23 Mbp. A contig of 12,269 bp including the DNA sequence of the B. algerae ribosomal transcription unit has been built from initial genomic sequences and the secondary structure of the large subunit rRNA constructed. The data obtained indicate that B. algerae should be an excellent parasitic model to understand genome evolution in relation to infectious capacity.

A case study of Desmozoon lepeophtherii infection in farmed Atlantic salmon associated with gill disease, peritonitis, intestinal infection, stunted growth, and increased mortality.

BACKGROUND: In September 2008, a disease outbreak characterized by acute, severe gill pathology and peritonitis, involving the gastrointestinal tract, was observed in an Atlantic salmon (Salmo salar L.) farm in north-western Norway. During subsequent sampling in November 2008 and January 2009, chronic proliferative gill inflammation and peritonitis was observed. Cumulative mortalities of 5.6-12.8% and severe growth retardation were observed. Routine diagnostic analysis revealed no diseases known to salmon at the time, but microsporidian infection of tissues was observed. METHODS: To characterize the disease outbreak, a combination of histopathology, in situ hybridization (ISH), chitin, calcofluor-white (CFW) staining, and real-time PCR were used to describe the disease progression with visualization of the D. lepeophtherii stages in situ. RESULTS: The presence of the microsporidian Desmozoon lepeophtherii was confirmed with real-time PCR, DNA sequencing and ISH, and the parasite was detected in association with acute lesions in the gills and peritoneum. ISH using a probe specific to small subunit 16S rRNA gene provided an effective tool for demonstrating the distribution of D. lepeophtherii in the tissue. Infection in the peritoneum seemed localized in and around pre-existing vaccine granulomas, and in the gastrointestinal walls. In the heart, kidney and spleen, the infection was most often associated with mononuclear leucocytes and macrophages, including melanomacrophages. Desmozoon lepeophtherii exospores were found in the nuclei of the gastrointestinal epithelium for the first time, suggesting a role of the gastrointestinal tract in the spread of spores to the environment. CONCLUSIONS: This study describes the progression of D. lepeophtherii disease outbreak in an Atlantic salmon farm without any other known diseases present. Using different methods to examine the disease outbreak, new insight into the pathology of D. lepeophtherii was obtained. The parasite was localized in situ in association with severe tissue damage and inflammation in the gills, peritoneal cavity and in the gastrointestinal (GI) tract that links the parasite directly to the observed pathology.

Description of a xenoma-inducing microsporidian, Microgemma tincae n. sp., parasite of the teleost fish Symphodus tinca from Tunisian coasts.

A xenoma-inducing microsporidian species was found to infect the liver of the teleost fish, peacock wrasse Symphodus (Crenilabrus) tinca. Minimal estimates of the prevalence of the parasite in fishes caught along Tunisian coasts were as high as 43 % for Bizerte samples (over 2 yr) and 72% for Monastir samples (over 3 yr). Developmental stages were dispersed within a xenoma structure that was bounded only by the plasma membrane of the hypertrophic host cell. Ultrastructural features support allocation to the genus Microgemma Ralphs and Matthews, 1986. Meronts were multinucleate plasmodia and were surrounded by rough endoplasmic reticulum (RER) of the host cell. Merogonic plasmodia developed into sporogonic plasmodia, with loss of the RER interface. Sporogony was polysporoblastic. Ovocylindrical spores (3.6 x 1.2 microm) harbored a lamellar polaroplast and a polar tube that was coiled 9 times. Spore features and host specificity led us to propose a new species, Microgemma tincae. The conversion of M. tincae xenomas into well-visible cyst structures or granulomas reflected an efficient host response involving the infiltration of phagocytic cells, degradation of various parasite stages and formation of a thick fibrous wall. The small subunit rDNA gene of M. tincae was partially sequenced. Phylogenetic analysis confirms the placement within the family Tetramicriidae represented by the genera Tetramicra and Microgemma.

Chromosomal composition of the genome in the monomorphic diplokaryotic microsporidium Paranosema grylli: analysis by two-dimensional pulsed-field gel electrophoresis.

The molecular karyotype of Paranosema grylli Sokolova, Seleznev, Dolgikh et Issi, 1994, a monomorphic diplokaryotic microsporidium, comprises numerous bright and faint bands of nonstoichiometric staining intensity. Restriction analysis of chromosomal DNAs by "karyotype and restriction display" 2-D PFGE has demonstrated that the complexity of molecular karyotype of P. grylli is related to the pronounced length polymorphism of-homologous chromosomes. The background of this phenomenon is discussed in the context of ploidy state, reproductive strategy and population structure in this microsporidium. We propose that the remarkable size variation between homologous chromosomes in P. grylli may be a consequence of ectopic recombination at the chromosome extremities.

Investigations into microsporidian methionine aminopeptidase type 2: a therapeutic target for microsporidiosis.

The Microsporidia have been reported to cause a wide range of clinical diseases particularly in patients that are immunosuppressed. They can infect virtually any organ system and cases of gastrointestinal infection, encephalitis, ocular infection, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles such as albendazole are active against many species of Microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin, ovalicin and their analogues have been demonstrated to have antimicrosporidial activity in vitro and in animal models of microsporidiosis. Fumagillin has also been demonstrated to have efficacy in human infections due to E. bieneusi. Fumagillin is an irreversible inhibitor of methionine aminopeptidase type 2 (MetAP2). Homology cloning employing the polymerase chain reaction was used to identify the MetAP2 gene from the human pathogenic microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Brachiola algerae and E. bieneusi. The full-length MetAP2 coding sequence was obtained for all of the Encephalitozoonidae. Recombinant E. cuniculi MetAP2 was produced in baculovirus and purified using chromatographic techniques. The in vitro activity and effect of the inhibitors bestatin and TNP-470 on this recombinant microsporidian MetAP2 was characterized. An in silico model of E. cuniculi MetAP2 was developed based on crystallographic data on human MetAP2. These reagents provide new tools for the development of in vitro assay systems to screen candidate compounds for use as new therapeutic agents for the treatment of microsporidiosis.

Close genotypic relationship between Enterocytozoon bieneusi from humans and pigs and first detection in cattle.

The reservoirs and the routes of transmission of Enterocytozoon bieneusi are still unknown. In humans, it is the most commonly found microsporidial species. It has also been found repeatedly in pigs, too. The first detection of E. bieneusi in cattle is reported herein. Two distinct genotypes were characterized and compared with 4 other genotypes from humans, 6 from pigs, and 1 from a cat. From these 13 E. bieneusi genotypes known to date, 25 polymorphic sites could be identified in the internal transcribed spacer of the rRNA gene. The spectrum of polymorphisms within and between each of the 4 host species indicates a close relationship between E. bieneusi strains from humans and pigs, whereas those from cattle are more distantly related. The data suggest the absence of a transmission barrier between pigs and humans for this pathogen.

A new intranuclear microsporidium, Enterospora nucleophila n. sp., causing an emaciative syndrome in a piscine host (Sparus aurata), prompts the redescription of the family Enterocytozoonidae.

The presence of a new microsporidium is believed to be responsible for an emaciative syndrome observed in farmed gilthead sea bream (Sparus aurata) from different facilities along the Spanish coast. Infected fish were approximately half the average weight and significant mortality was attributed to the condition in some facilities. Clinical signs included anorexia, cachexia and pale internal organs. The microsporidium was found mainly in the intestinal mucosa and occasionally in the submucosa. Morphological, histopathological, ultrastructural and molecular phylogenetic studies were conducted to characterise this organism. This microsporidium undergoes intranuclear development in rodlet cells and enterocytes, and cytoplasmic development mainly in enterocytes and macrophages. The nucleus-infecting plasmodium contains several diplokarya and displays polysporous development which occurs without an interfacial envelope. In the host cell cytoplasm, the parasite develops within a membrane-bound matrix. In both infection locations, the polar tube precursors appear as disks, first with lucent centres, then as fully dense disks as they fuse to form the polar filament, all before division of the plasmodium into sporoblasts. Up to 16 intranuclear spores result from the sporogonic development of a single plasmodium, whereas more than 40 spores result from several asynchronous reproductive cycles in the cytoplasmic infection. Fixed spores are ellipsoidal and diplokaryotic, with five to six coils of an isofilar polar filament in a single row. ssrDNA-based molecular phylogenetic inference places this parasite as a sister clade to crustacean-infecting species of the Enterocytozoonidae and closer to Enterocytozoon bieneusi than to other fish-infecting microsporidians presenting intranuclear development, i.e. Nucleospora, Paranucleospora and Desmozoon. Our studies result in the erection of a new species, Enterospora nucleophila, within the family Enterocytozoonidae, and the description of this family is amended accordingly to accommodate the features of known species assigned to it. Severe histopathological damage occurs in intense infections and this microsporidian is considered a serious emerging threat in sea bream production.

Secretion of Antonospora (Paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes.

Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/ß-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/ß-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs.