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Most eukaryotic cells have a plasma membrane with glycoproteins on the outer leaflet and cytoplasmic proteins on the inner leaflet. However, the microsporidians examined have a peculiar outer membrane with cytoplasmic proteins on the surface. His study was to determine if this is true and identify the presence of key cytoplasmic proteins on the exterior of the sporoplasm and spore stages. Specific probes including GFP-labeled genes, antibodies, and electron microscopy were applied to studies on the sporoplasm and spore stages of different microsporidian species. Cytoplasmic membrane proteins cadherin, intermediate filaments, dynactin p150Glued and tubulin were identified on the outer leaflet of the exterior membrane of the sporoplasm or spore stages of Spraguea americanus, Anncaliia algerae, Ameson michaelis and Thelohania sp. The invasive sporoplasm stage of microsporidians acquires an outer membrane from a cytoplasmic organelle and it has been shown that the outside surface of this membrane bears cytoplasmic proteins. This membrane retains these cytoplasmic proteins even on the spore stage. For an intracellular parasite that locates directly within the cytoplasm of a host cell, the presence of these cytoplasmic proteins on the surface of the parasite appears to have a significant effect on the host’s response.

As one of the core framework proteins of nuclear pore complex (NPC), nucleoporin Nupl70 acts as a structural adapter between the nucleolus and nuclear pore membrane and maintains the stability of NPC structure through interaction with other proteins. In this study, we identified a Nup170 protein in the microsporidian Nosema bombycis for the first time and named it as NbNup170. Secondary structure prediction showed that the NbNup170 contains α-helices and random coils. The three-dimensional structure of NbNup170 is elliptical in shape. Phylogenetic analysis based on the Nup170 and homologous sequences showed that N. bombycis clustered together with Vairimorpha ceranae and Vairimorpha apis. The immunofluorescence localization results showed that the NbNup170 was located on the plasma membrane of the dormant spore and transferred to the surface of sporoplasm in a punctate pattern when the dormant spore has finished germination, and that NbNup170 was distributed on the nuclear membrane and both sides of the nuclei of early proliferative phase, and only on the nuclear membrane during sporogonic phase in the N. bombycis. qPCR analysis showed that the relative expression level of NbNup170 maintained at a low level from 30 to 78 h post-infection with N. bombycis, then reached the highest at 102 h, while that of NbNup170 was repressed at a very low level throughout its life cycle by RNA interference. These results suggested that NbNup170 protein is involved in the proliferative phase and active during the sporogonic phase of N. bombycis.

The present paper describes a novel species of Myxobolus parasitizing the gill filaments of the largescale mullet, Planiliza macrolepis from Cochin backwaters, Kerala, India. The parasite develops in the gill filaments; plasmodia elongated, milky white, measured 1.37 – 2.18 (1.78 ± 0.35) mm x 0.07-0.12 (0.10 ± 0.02) mm in size. Mature myxospores ovoid in valvular view, biconvex in sutural view with smooth shell valves and measured 6.24 - 7.02 (6.63 ± 0.23) × 5.01 - 6.18 (5.68 ± 0.25) μm in size. Polar capsules equal, oval with pointed anterior ends, 3.07 – 3.58 (3.33 ± 0.12) × 1.68 – 2.42 (2.09 ± 0.18) μm in size. Polar filaments with 4 coils, measured 29.61 ± 4.75 μm in length when extruded. Sporoplasm binucleate with a rudimentary nucleus and a vacuole. A comparison with related Myxobolus species revealed significant morphological & morphometric differences. In BLASTN and genetic distance analysis, the present parasite showed high divergence with other myxosporean sequences, indicating its molecular uniqueness. In Maximum Likelihood and Bayesian Interference analysis, the present species stands out with M. ramadus as sister branch within the Myxobolus clade. In infected gill filaments, the plasmodia caused swelling/deformation, compression of lamellae and reduction in respiratory surface area. Three of 222 P. macrolepis screened were infected, indicating a prevalence of 1.3%. Considering the morphological, morphometric, molecular and phylogenetic differences with the previously described species of myxosporeans, along with the dissimilarities in host and geographical locations, the present parasite is treated as a new species and the name Myxobolus cochinensis n. sp. is proposed.

Background Nosema bombycis is a unicellular eukaryotic pathogen of the silkworm, Bombyx mori, and is an economic and occupational hazard in the silkworm industry. Because of its long incubation period and horizontal and vertical transmission, it is subject to quarantine measures in sericulture production. The microsporidian life-cycle includes a dormant extracellular phase and intracellular proliferation phase, with the proliferation period being the most active period. This latter period lacks spore wall protection and may be the most susceptible stage for control. Methods In order to find suitable target for the selective breeding of N. bombycis-resistant silkworm strains, we screen highly expressed membrane proteins from the transcriptome data of N. bombycis. The subcellular localization of the candidate protein was verified by Indirect immunofluorescence analysis (IFA) and immunoelectron microscopy (IEM), and its role in N. bombycis proliferation was verified by RNAi. Results The N. bombycis protein (NBO_76g0014) was identified as a transmembrane protein and named NbTMP1. It is homologous with hypothetical proteins NGRA_1734 from Nosema granulosis. NbTMP1 has a transmembrane region of 23 amino acids at the N-terminus. Indirect immunofluorescence analysis (IFA) results suggest that NbTMP1 is secreted on the plasma membrane as the spores develop. Western blot and qRT-PCR analysis showed that NbTMP1 was expressed in all developmental stages of N. bombycis in infected cells and in the silkworm midgut. Downregulation of NbTMP1 expression resulted in significant inhibition of N. bombycis proliferation. Conclusions We confirmed that NbTMP1 is a membrane protein of N. bombycis. Reduction of the transcription level of NbTMP1 significantly inhibited N. bombycis proliferation, and this protein may be a target for the selective breeding of N. bombycis-resistant silkworm strains. Graphical Abstract

Background Microsporidians are opportunistic pathogens with a wide range of hosts, including invertebrates, vertebrates and even humans. Microsporidians possess a highly specialized invasion structure, the polar tube. When spores encounter an appropriate environmental stimulation, the polar tube rapidly everts out of the spore, forming a 50–500 µm hollow tube that serves as a conduit for sporoplasm passage into host cells. The polar tube is mainly composed of polar tube proteins (PTPs). So far, five major polar tube proteins have been isolated from microsporidians. Nosema bombycis, the first identified microsporidian, infects the economically important insect silkworm and causes heavy financial loss to the sericulture industry annually. Results A novel polar tube protein of N. bombycis (NbPTP6) was identified. NbPTP6 was rich in histidine (H) and serine (S), which contained a signal peptide of 16 amino acids at the N-terminus. NbPTP6 also had 6 potential O-glycosylation sites and 1 potential N-glycosylation site. The sequence alignment analysis revealed that NbPTP6 was homologous with uncharacterized proteins from other microsporidians (Encephalitozoon cuniculi, E. hellem and N. ceranae). Additionally, the NbPTP6 gene was expressed in mature N. bombycis spores. Indirect immunofluorescence analysis (IFA) result showed that NbPTP6 is localized on the whole polar tube of the germinated spores. Moreover, IFA, enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays results revealed that NbPTP6 had cell-binding ability. Conclusions Based on our results, we have confirmed that NbPTP6 is a novel microsporidian polar tube protein. This protein could adhere with the host cell surface, so we speculated it might play an important role in the process of microsporidian infection.

Ceratomyxa mennani n. sp. is a new coelozoic Ceratomyxa species found in the gallbladder of Epinephelus marginatus from the Gulf of Tunis, Tunisia. Mature plasmodia were disporic, ovoid in shape measuring 9-12 μm in width and 11-14 μm in length. Mature myxospores were slightly crescent-shaped with almost straight posterior margin, measuring 5.8 ± 0.2 (5.4-6.1) μm in length and 12.7 ± 0.3 (11.9-13.0) μm in thickness. The two valves were unequal with rounded ends. Polar capsules were spherical, equal in size with 2.1 ± 0.2 (1.9-2.6) μm in diameter. The binucleated sporoplasm filled the entire cavity of the myxospore. Molecular analysis of SSU rDNA sequences indicated that C. mennani n. sp. was distinct from all other Ceratomyxa sequences in GenBank. Phylogenetic analysis revealed that C. mennani n. sp. clustered with Ceratomyxa species infecting Epinephelinae fishes. Seasonal prevalence of infection over one year was significantly higher in winter and the lowest in autumn. This is the third report of Ceratomyxa species infecting the gallbladder of Epinephelus marginatus from Tunisia and the first study to include molecular data.

Myxozoans of the family Ceratomyxidae are common coelozoic parasites of marine, anadromous, and freshwater fish, and may also be found, less frequently, parasitizing the tissue of these hosts. The diversity and ecology of the freshwater species of the genus Ceratomyxa have been poorly investigated, leading to a knowledge gap that restricts the understanding of the distribution and prevalence of this group of parasites. In the present study, parasites were found inside vermiform plasmodia, characterised by oscillatory movements in the characiform species Hemiodus unimaculatus. The crescent-shaped and elongated spores, perpendicular to the suture line, have a mean length of 28.9 ± 2.7 μm and width of 2.6 ± 0.1 μm, with two symmetrical oval polar capsules, 1.9 ± 0.3 μm in length and 1.7 ± 0.2 μm in width, containing polar filaments with three or four coils, located near the central suture, with symmetrical lateral elongations 14.3 ± 1.1 μm in length and binucleate amoeboid sporoplasm. The integrated comparative analysis of the morphological characteristics and partial SSU rRNA sequences supported the identification of a new species of coelozoic Ceratomyxa, found in the gallbladder of H. unimaculatus, from the Tocantins basin, in the municipalities of Estreito and Imperatriz in eastern Brazilian Amazonia.The new species was denominated Ceratomyxa fonsecai n. sp.

All Microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore, transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200nm, noticeably larger than fixed-sectioned, or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5–6nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.

ATP-binding cassette (ABC) transporters comprise the largest family of transmembrane proteins and are found in all domains of life. The ABCs are involved in a variety of biological processes and as exporters play important roles in multidrug resistance. However, the ABC transporters have not been addressed in microsporidia, which are a very large group of obligate intracellular parasites that can infect nearly all animals, including humans. Here, a total of 234 ABC transporters were identified from 18 microsporidian genomes and classified into five subfamilies, including 74 ABCBs, 2 ABCCs, 18 ABCEs, 15 ABCFs, 102 ABCGs and 23 uncategorized members. Two subfamilies, ABCA and ABCD, are found in most organisms, but lost in microsporidia. Phylogenetic analysis indicated that microsporidian ABCB and ABCG subfamilies expanded by recent gene duplications, which resulted in the two largest subfamilies in microsporidia. Functional analysis via qRT-PCR and Western blotting revealed that NoboABCG1.1, an ABCG member of Nosema bombycis, is expressed in mature spores and up-regulated from 1 dpi to 6 dpi in infected silkworm midgut. IFA and IEM analysis showed that NoboABCG1.1 is localized on the plasma membrane of the sporoplasm, meront and mature spore. The propagation of N. bombycis was significantly inhibited after the RNAi of NoboABCG1.1 expression, indicating that NoboABCG1.1 is important to the pathogen proliferation. In conclusion, our study uncovered that the ABCs evolved during microsporidia adaption to intracellular parasitism and play important roles in the pathogen development.

Two new species of Myxobolus Butschli, 1882 (Bivalvulida: Myxobolidae) are described from the gill of the black redhorse (Moxostoma duquesnei [Leueur][Cypriniformes: Catostomidae]) from the Little Tennessee River Basin, North Carolina, United States. Myxobolus branchiofilum n. sp. infects lumen of the lamellar arterioles and Myxobolus branchiopecten n. sp. infects the bone and cartilage at the tip of the gill rakers. They differ from all congeners by a combination of myxospore dimensions and the presence or absence of an iodinophilic vacuole in the sporoplasm, mucous envelope, intercapsular process, and sutural markings. A phylogenetic analysis of the small subunit ribosomal DNA recovered M. branchiopectin sister to Myxobolus sp. (AF378343) in a clade composed of 6 species of Myxobolus, which infect predominately cypriniform intermediate hosts. Myxobolus branchiofilum was recovered sister to Myxobolus ictiobus Rosser, Griffin, Quiniou, Alberson, Woodyard, Mischker, Greenway, Wise & Pote, 2016 in a clade composed of 8 species of Myxobolus, which predominately infect catostomid intermediate hosts. Histological sections of infected gill revealed intra-lamellar plasmodia of M. branchiofilum in the lumen of the lamellar arterioles and foci of M. branchiopecten developing in the bone and cartilage of the gill raker tip. These are the first myxozoans reported from the black redhorse. Given that these two new species are morphologically congeneric but recovered in distantly related clades, we discuss the persistent issue of myxobolid genera paraphyly/polyphyly.

Answer: d. Spores undergo gemination resulting in extrusion of polar tubule into the host cell. Via the polar tubule, the infective sporoplasm is released into the host cell, which undergoes various stages of development. Answer: a. Electron microscopy. Transmission electron microscopy helps in

The sicklefin redhorse, Moxostoma sp. (Cypriniformes: Catostomidae), is an innominate imperiled catostomid endemic to the Hiwassee and Little Tennessee river basins, which has been restricted to a few tributaries of these systems by impoundments. During collections to propagate sicklefin redhorse for reintroduction, a myxozoan, described herein, was observed infecting sicklefin redhorse in the Little Tennessee River Basin, North Carolina. Myxobolus naylori Ksepka et Bullard sp. n. infects the stratum spongiosum covering the scales of sicklefin redhorse. Myxospores of the new species differ from all congeners by the combination of having a mucous envelope, intercapsular process, and sutural markings as well as lacking an iodinophilic vacuole in the sporoplasm. A phylogenetic analysis of the 18S rDNA gene recovered the new species in a polytomy with Myxobolus marumotoi Li et Sato, 2014 and a clade comprised of species of Myxobolus Butschli, 1882; Thelohanellus Kudo, 1933, and Dicauda Hoffman et Walker, 1973. Histological sections of infected sicklefin redhorse skin revealed myxospores within a plasmodium in the stratum spongiosum dorsal to scales, encapsulated in collagen fibres, and associated with focal erosion of scales directly beneath the plasmodium; in some instances, the scale was perforated by the plasmodium. The specificity of the new species to sicklefin redhorse may make it a useful biological tag to differentiate sicklefin redhorse from morphologically similar species. The new species is the first parasite reported from sicklefin redhorse, a species of concern to the United States Fish and Wildlife Service. No species of Myxobolus has been reported from species of Moxostoma in the Southeast United States. As it was observed that Myxobolus minutus Rosser, Griffin, Quiniou, Alberson, Woodyard, Mischker, Greenway, Wise et Pote, 2016 is a primary junior homonym of Myxobolus minutus Nemeczek, 1911, we propose the replacement name Myxobolus diminutus (Rosser, Griffin, Quiniou, Alberson, Woodyard, Mischker, Greenway, Wise et Pote, 2016).

Microsporidia are found worldwide and both vertebrates and invertebrates can serve as hosts for these organisms. While microsporidiosis in humans can occur in both immune competent and immune compromised hosts, it has most often been seen in the immune suppressed population, e.g., patients with advanced HIV infection, patients who have had organ transplantation, those undergoing chemotherapy, or patients using other immune suppressive agents. Infection can be associated with either focal infection in a specific organ (e.g., keratoconjunctivitis, cerebritis, or hepatitis) or with disseminated disease. The most common presentation of microsporidiosis being gastrointestinal infection with chronic diarrhea and wasting syndrome. In the setting of advanced HIV infection or other cases of profound immune deficiency microsporidiosis can be extremely debilitating and carries a significant mortality risk. Microsporidia are transmitted as spores which invade host cells by a specialized invasion apparatus the polar tube (PT). This review summarizes recent studies that have provided information on the composition of the spore wall and PT, as well as insights into the mechanism of invasion and interaction of the PT and spore wall with host cells during infection.

Once awoken from dormancy, the cellular matter of microsporidia is delivered directly into the host cell cytoplasm through the polar tube. This means that the microsporidia are difficult to study biologically in their active state without a contaminating signal from the host cell. Sporoplasm is a cell type of microsporidia in vitro, but relatively little attention has been paid to the sporoplasm in the past 150 years due to a lack of an effective separation method. Nosema bombycis, the first reported microsporidium, is a type of obligate intracellular parasite that infects silkworms and can be induced to germinate in alkaline solution in vitro. We successfully separated the N. bombycis sporoplasm in vitro, and the morphological and structural characteristics were investigated. These results provide important insight into the biology and pathogenesis of microsporidia and potentially provide a possible strategy for genetic manipulation of microsporidia targeting the sporoplasm., Microsporidia are obligate intracellular parasites that infect a wide variety of host organisms, including humans. The sporoplasm is the initial stage of microsporidian infection and proliferation, but its morphological and molecular characteristics are poorly understood. In this study, the sporoplasm of Nosema bombycis was successfully isolated and characterized after the induction of spore germination in vitro. The sporoplasm was spherical, 3.64 ± 0.41 μm in diameter, had the typical two nuclei, and was nonrefractive. Scanning and transmission electron microscopy analyses revealed that the sporoplasm was surrounded by a single membrane, and the cytoplasm was usually filled with relatively homogeneous granules, possibly ribosomes, and contained a vesicular structure comprising a concentric ring and coiled tubules. Propidium iodide staining revealed that the sporoplasm membrane showed stronger membrane permeability than did the cell plasma membrane. Transmission electron microscopy (TEM) revealed that the sporoplasm can gain entry to the host cell by phagocytosis. Transcriptome analysis of mature spores and sporoplasms showed that 541 significantly differentially expressed genes were screened (adjusted P value [Padj] < 0.05), of which 302 genes were upregulated and 239 genes were downregulated in the sporoplasm. The majority of the genes involved in trehalose synthesis metabolism, glycolysis, and the pentose phosphate pathway were downregulated, whereas 10 transporter genes were upregulated, suggesting that the sporoplasm may inhibit its own carbon metabolic activity and obtain the substances required for proliferation through transporter proteins. This study represents the first comprehensive and in-depth investigation of the sporoplasm at the morphological and molecular levels and provides novel insights into the biology of microsporidia and their infection mechanism. IMPORTANCE Once awoken from dormancy, the cellular matter of microsporidia is delivered directly into the host cell cytoplasm through the polar tube. This means that the microsporidia are difficult to study biologically in their active state without a contaminating signal from the host cell. Sporoplasm is a cell type of microsporidia in vitro, but relatively little attention has been paid to the sporoplasm in the past 150 years due to a lack of an effective separation method. Nosema bombycis, the first reported microsporidium, is a type of obligate intracellular parasite that infects silkworms and can be induced to germinate in alkaline solution in vitro. We successfully separated the N. bombycis sporoplasm in vitro, and the morphological and structural characteristics were investigated. These results provide important insight into the biology and pathogenesis of microsporidia and potentially provide a possible strategy for genetic manipulation of microsporidia targeting the sporoplasm.

A new microscopic cnidarian is described, infecting five of thirty (16.6%) specimens of Corydoras schwartzi caught in the Purus River, State of Amazonas, Brazil. Histological analysis showed that cyst development occurred in the serosa layer of the intestine. Mature myxospores are ovoid in body shape in frontal view, 22.4 ± 0.3 μm in total length and 16.3 ± 0.1 μm in width. Internally, two aubergine-shaped, elongate symmetrical polar capsules occupy more than half the length of the spore, 14.3 ± 0.2 μm in length and 6.5 ± 0.1 μm in width. Ultrastructural analysis provided evidence of five polar filament coils inside the polar capsule and binucleated sporoplasm containing a moderate number of sporoplasmosomes. The valvogenic cells abutting each other form a sutural ridge and frequently a thin layer of homogeneous material separates the cells. The outer surface of the myxospore valves is smooth, with no evidence of formation of ridges in the valves. Immature myxospores in various stages of development were observed. This study is the first report of a myxosporean parasitizing C. schwartzi and the first report of a myxosporean infection in the intestine of an ornamental fish from South America.

Microsporidia are important opportunistic human pathogens in immune-suppressed individuals, such as those with HIV/AIDS and recipients of organ transplants. The sporoplasm is critical for establishing microsporidian infection. Despite the biological importance of this structure for transmission, there is limited information about its structure and composition that could be targeted for therapeutic intervention. Here, we identified a novel E. hellem sporoplasm surface protein, EhSSP1, and demonstrated that it can bind to host cell mitochondria via host VDAC. Our data strongly suggest that the interaction between SSP1 and VDAC is important for the association of mitochondria with the parasitophorous vacuole during microsporidian infection. In addition, binding of SSP1 to the host cell is associated with the final steps of invasion in the invasion synapse., Microsporidia are opportunistic intracellular pathogens that can infect a wide variety of hosts ranging from invertebrates to vertebrates. During invasion, the microsporidian polar tube pushes into the host cell, creating a protective microenvironment, the invasion synapse, into which the sporoplasm extrudes. Within the synapse, the sporoplasm then invades the host cell, forming a parasitophorous vacuole (PV). Using a proteomic approach, we identified Encephalitozoon hellem sporoplasm surface protein 1 (EhSSP1), which localized to the surface of extruded sporoplasms. EhSSP1 was also found to interact with polar tube protein 4 (PTP4). Recombinant EhSSP1 (rEhSSP1) bound to human foreskin fibroblasts, and both anti-EhSSP1 and rEhSSP1 caused decreased levels of host cell invasion, suggesting that interaction of SSP1 with the host cell was involved in invasion. Coimmunoprecipitation (Co-IP) followed by proteomic analysis identified host cell voltage-dependent anion channels (VDACs) as EhSSP1 interacting proteins. Yeast two-hybrid assays demonstrated that EhSSP1 was able to interact with VDAC1, VDAC2, and VDAC3. rEhSSP1 colocalized with the host mitochondria which were associated with microsporidian PVs in infected cells. Transmission electron microscopy revealed that the outer mitochondrial membrane interacted with meronts and the PV membrane, mitochondria clustered around meronts, and the VDACs were concentrated at the interface of mitochondria and parasite. Knockdown of VDAC1, VDAC2, and VDAC3 in host cells resulted in significant decreases in the number and size of the PVs and a decrease in mitochondrial PV association. The interaction of EhSSP1 with VDAC probably plays an important part in energy acquisition by microsporidia via its role in the association of mitochondria with the PV.

Two myxosporean species of the genus Sphaeromyxa were isolated from the gallbladders of marine fish in the South China Sea. Sphaeromyxa scorpaena n. sp. was collected from Scorpaenodes albaiensis Evermann and Seale, 1907. The mature myxospores were arcuate-shaped with tapered to pointed ends, and a length of 14.1 ± 0.7 (13.8–15.1) μm and a width of 5.2 ± 0.3 (4.9–5.8) μm. The polar capsules (PCs) were pyriform with a length of 3.2 ± 0.2 (3.1–3.5) μm and a width of 1.6 ± 0.1 (1.4–1.8) μm, and containing ribbon-like polar filaments irregularly folded 1.5–2.5 turns. Molecular characteristics and phylogenetic analysis based on 18S rDNA as well as morphological comparison confirmed that S. scorpaena n. sp. was a previously undescribed species. Sphaeromyxa theraponi, isolated from Terapon jarbua Forsskal, 1775, was reported for the first time from the South China Sea. The mature myxospores were slightly arched, tapering to bluntly rounded ends, with a length of 17.3 ± 0.9 (15.5–19.4) μm and a width of 4.8 ± 0.3 (4.1–5.3) μm. A sporoplasm was situated in the space between PCs in the myxospore. The PCs were pyriform, which contained ribbon-like polar filaments irregularly folded by 2–3 turns, with a length of 7.0 ± 0.5 (5.8–8.1) μm and a width of 2.6 ± 0.2 (2.2–3.0) μm. Our morphological and phylogenetic analyses suggest that the pointed ends of S. scorpaena n. sp. might be a secondarily acquired characteristic rather than an ancestral trait.

The present study attempted sequencing the 18S rRNA gene of Myxoboluscatmrigalae infecting the gill lamellae of carp, Cirrhinusmrigala and compared its genetic homology and phylogenetic characteristics with 18S rRNA genes of other Myxobolus spp. The infected fish had up to 3 small, creamy white plasmodia per gill filament with 30-50 spores each. The spore size was 17.90 ± 0.70 × 7.40 ± 0.40 μm. The sporoplasm contained two large nuclei of size 0.57 ± 0.09 μm and no iodinophilous vacuole. The DNA sequence of M.catmrigalae was clustered phylogenetically with other Myxobolus spp. infecting the gills of cyprinids available in GenBank, which showed 77-87 % homogeneity. On the phylogenetic tree, M.catmrigalae (KC933944) was clustered with M.pavlovskii (HM991164) infecting the gill lamellae of silver carp, Hypophthalmichthysmolitrix. The species most closely related to M.catmrigalae in GenBank was M.pavlovskii (AF507973) infecting the gill lamellae of big head carp, Aristichthysnobilis with 87 % homogeneity. This is the first report on molecular characterization of gill lamellae infecting M. catmrigalae.

Microsporidia are obligate intracellular parasites and cannot be cultured in vitro, which limits the use of current genetic engineering technologies on this pathogen. We isolated sporoplasms of Nosema bombycis to attempt to culture the pathogen in vitro. Cell-free medium was designed and successfully maintained the sporoplasms for 5 days. The sporoplasms were able to absorb ATP from the medium and DNA replicated during cultivation, although there was not a significant change in morphology and number of sporoplasms. Our study provides a strategy for in vitro cultivation and genetic manipulation of microsporidia. .

Nosema bombycis, the first identified microsporidium, causes heavy losses to the sericulture industry in China. During infection, microsporidia discharge a long and hollow polar tube, which delivers the sporoplasm into host cells. Polar tube protein 1 was the major component on the polar tube. Previously, we expressed the polar tube protein 1 from Nosema bombycis (NbPTP1) intercellularly in Drosophila S2 cells. Here, the microsporidian protein was expressed in Lepidopteran Sf9 cells. During heterologous expression, NbPTP1 protein was secreted and glycosylated. Microsporidian proliferation decreased in NbPTP1-expressing Sf9 cells. This confirms that NbPTP1 protein can interact with the host cell membrane receptor protein to facilitate microsporidian invasion.

AimsTo report the clinical manifestations, ultrastructure and evaluate the efficacy of therapeutic lamellar keratectomy (TLK) and penetrating keratoplasty (PK) for microsporidial stromal keratitis (MSK).MethodsFourteen MSK cases between 2009 and 2018 were recruited. Each patient’s clinical presentation, light microscopy, histopathology, PCR and electron microscopy (EM) of corneal samples were reviewed.ResultsThe patients were 70.0±4.7 years old (average follow-up, 4.5 years). Time from symptoms to presentation was 10.6±13.0 weeks. The corneal manifestations were highly variable. Corneal scrapings revealed Gram stain positivity in 12 cases (85.7%) and modified Ziehl-Neelsen stain positivity in 9 (64.3%). Histopathology revealed spores in all specimens, while sequencing of small subunit rRNA-based PCR products identified Vittaforma corneae in 82% of patients. EM demonstrated various forms of microsporidial sporoplasm in corneal keratocytes. All patients were treated with topical antimicrobial agents or combined with oral antiparasitic medications for >3 weeks. As all patients were refractory to medical therapy, they ultimately underwent surgical intervention (TLK in 7, PK in 6 and 1 received TLK first, followed by PK). Postoperatively, the infection was resolved in 78.6% of the patients. Nevertheless, a high recurrence rate (21.4%) was noted during 3-year follow-up, with only two patients retained a final visual acuity ≥20/100.ConclusionMSK usually presents with a non-specific corneal infiltration refractory to antimicrobial therapy. The diagnosis relies on light microscopic examinations on corneal scrapings and histopathological analyses. Surgical intervention is warranted by limiting the infection; however, it was associated with an overall poor outcome.

This paper describes 2 new myxosporean species, Henneguya lepturus sp. nov. and Thelohanellus lepturus sp. nov., simultaneously infecting the brain and spinal cord of Hypopygus lepturus Hoedeman, 1962 (Teleostei, Hypopomidae) from the Brazilian Amazon (Roraima State). Several spherical cysts of varying dimensions (up to 135 µm) were microscopically observed. The myxospores of H. lepturus sp. nov. measured 25.8 µm in total length, having an ellipsoidal body (12.4 × 6.4 × 2.2 µm) and 2 equal tapering tails (13.4 µm in length). Each of the 2 pyriform polar capsules measured 4.4 × 1.6 µm and possessed a polar filament coiled in 8-9 turns. The myxospores of T. lepturus sp. nov. were pyriform, formed by 2 equal valves (17.7 × 9.1 × 4.3 µm) surrounding a single polar capsule (10.9 × 3.5 µm) that had a coiled polar filament with 13-16 turns and a binucleated sporoplasm that contained several circular sporoplasmosomes. Molecular analysis of the small subunit (SSU) rRNA gene sequences of these 2 species were in agreement with the taxonomic classification derived from the ultrastructure of the myxospores. Histopathology of the host tissue showed degradation of the myelinated axons surrounding the cysts of both species, with the hosts displaying behavioural changes and erratic movements when observed in an aquarium.

Microsporidia are eukaryotic unicellular parasites that have been studied for more than 150 years. They are found throughout the world and are capable of infecting various invertebrate and vertebrate hosts. They can cause disease in both immune-compromised and immune-competent humans. In immune-compromised individuals, infections can be severe and often fatal. Microsporidia possess a unique, highly specialized invasion mechanism that involves a structure known as the polar tube as well as the spore wall. During spore germination, the polar tube rapidly discharges from the spore and deliver the sporoplasm into the host cell. Spores are the only stage of microsporidia that can survive outside of host cells. Since the first attempt to culture microsporidia in vitro in 1930s, their cultivation has served a critical role in the study and diagnosis of these parasites. In this chapter, we include methods on the cultivation, isolation, and cryopreservation of Encephalitozoon cuniculi, which can infect humans and provides a useful model for other microsporidia. These methods can also be utilized for the culture of Encephalitozoon hellem or Encephalitozoon intestinalis. © 2018 by John Wiley & Sons, Inc.

This study describes infection of intestinal smooth muscle in fringelip mullets Crenimugil crenilabis with Kudoa crenimugilis n. sp. Of 30 individuals sampled from the Red Sea off Saudi Arabia, 6 (20%) were infected. Ovoid plasmodia (279-412 × 157-295 µm) in the smooth muscle of the intestine were packed with only mature myxospores with 4 valves. Specifically, light and transmission electron microscopy revealed quadrate myxospores with 4 equal, rounded, spore valves uniting at thin delicate suture lines. The mature myxospores were 8 (7-9) µm long, 5.2 (5-6) µm thick and 7.8 (7-8) µm wide. The 4 polar capsules were equal-sized, elliptical to ovoid, and measured 5 (4-5) µm long and 2 (1.5-3) µm wide, possessing 2 filament coils. The sporoplasm was uninucleated and composed of a primary cell enveloping a secondary cell. The parasite had a significant histopathological impact since the developing plasmodia replaced normal muscle tissue and was associated with the myolysis of local muscle fibres and the inflammatory infiltration of lymphocytes and macrophages. The partial sequences of the 18S and 28S rDNA showed that K. crenimugilis n. sp. has the highest level of nucleotide similarity with K. ciliatae (98.46 and 94.11%, respectively) and K. cookii (97.51 and 92.11%, respectively), both of which have previously been reported from the intestines of their host fish. Phylogenetic analysis revealed that K. crenimugilis consistently clustered with these other 2 intestinal Kudoa species in a well-supported subclade, confirming the evaluative association between Kudoa species infecting the same organs.

Myxobolus absonusCellere et al., 2002 was originally described as having free cysts in the opercular cavity of the freshwater fish Pimelodus maculatus in Brazil. The present study provides a supplemental description of this parasite from the eye of its type host, with basis on morphological, ultrastructural, and molecular data of the SSU rRNA gene. The parasite formed spherical whitish cysts, which wall presented numerous microvilli that attached to the collagen layers of the corneal stroma. Myxospores were oval in valvular and sutural view, measuring 13.2 ± 0.4 μm in length, 8.5 ± 0.4 μm in width, and 6.6 ± 0.3 μm in thickness. Two asymmetric pyriform polar capsules were located at the anterior pole: the larger 6.2 ± 0.4 μm long and 3.6 ± 0.3 μm wide, containing a polar filament coiled in 6 turns; and the smaller 3.5 ± 0.3 μm long and 1.9 ± 0.1 μm wide, containing a polar filament coiled in 4 turns. At the posterior pole, the sporoplasm displayed two nuclei and numerous spherical sporoplasmosomes. Phylogenetic analysis using maximum likelihood, Bayesian inference and maximum parsimony revealed M. absonus clustering within a well-supported clade with poorly-resolved internal nodes, amongst the SSU rRNA sequences of other myxobolids that infect siluriform and characiform fish hosts, as well as the perciform-infecting species Myxobolus acanthogobii, Triangula percae and Cardimyxobolus japonensis. This clade appeared separated from the other clades comprising most of the species that infect siluriform and characiform hosts, showing that more than one myxobolid lineage evolved while parasitizing these taxonomic groups of fish.

© 2015 Elsevier Inc.Microsporidia are highly divergent fungi that are obligate intracellular pathogens of a wide range of host organisms. Here we review recent findings from the genome sequences of mosquito-infecting microsporidian species Edhazardia aedis and Vavraia culicis, which show large differences in genome size, although similar numbers of predicted genes. We also show a video of E. aedis polar tube firing, which is the dramatic mechanism used by microsporidia to deliver the germ cell (sporoplasm) into the host cell to initiate intracellular infection.

During a survey of myxosporean parasites of marine fishes from the Arabian Gulf in Saudi Arabia, spores of Ceratomyxa husseini n. sp. were found in the gall-bladders of 50 out of 148 specimens (33.8%) of the yellowfin hind Cephalopholis hemistiktos (Rüppell) (Perciformes: Serranidae). The rates of infection showed a seasonal fluctuation, with the highest prevalence in winter and the lowest in autumn. The mature spores appeared arched in frontal view with rounded valve ends and a slightly discriminated curved suture line and measured 8-9 × 14-18 (9 × 16) μm. The two polar capsules were spherical and equal in size, 4-5 (4.5) μm in diameter. The polar filament showed four turns obliquely to the longitudinal axis of the capsules and the sporoplasm filled half of the entire spore cavity. Partial sequences of the small subunit rRNA gene of C. husseini n. sp. showed percentage of identity with other species of Ceratomyxa ranging between 79.8 and 92.7%. The morphometric and molecular data, in association, confirmed that the present new species differs from all other Ceratomyxa spp. reported to date.

The myxosporean parasite Kudoa septempunctata has been isolated from cultured olive flounder (Paralichthys olivaceus) and was recently identified as a cause of food poisoning in humans. Since the sporoplasm plays an important role in causing diarrhea by invading intestinal cells, the specific factors affecting the release of sporoplasm from spores should be determined. Thus, we investigated the effect of digestive and serum enzymes, fetal bovine serum (FBS), temperature, and the role of glucose in cell culture media on the release of sporoplasm. Sporoplasm release was observed in the groups treated with FBS and media containing glucose. In addition, 1,10-phenanthroline inhibited the release of sporoplasm in the FBS medium. These results indicate that K. septempunctata uses glucose for releasing its sporoplasm and that zinc or metalloprotease is related to the release mechanism. The present study provides important information for the development of agents to prevent sporoplasm release and the consequent food poisoning caused by K. septempunctata.

The present paper describes a new species of Chloromyxum infecting the urinary bladder of the estuarine fish, Scatophagus argus, from the southwest coast of India. The parasite exhibited an overall prevalence of 41.93%; the prevalence is influenced by host size and seasons. Mature spores are subspherical, measure 9.40 ± 0.66 by 9.32 ± 0.87 μm, and are characterized by the presence of sutural and extra-sutural ridges, binucleated sporoplasm, and a pair of caudal extensions. Four pyriform, unequal polar capsules with raised polar filament discharge pores and ribbon-like polar filaments are present. Polar filament coils numbered four to five in large polar capsules and three in small polar capsules. Pansporoblast is irregular with granulated cytoplasm and has fine villosites on its surface. Plasmodia are spherical/irregular with monosporic and polysporic forms. In molecular and phylogenetic analysis, the myxosporean stands out with a high bootstrap value and was positioned as a sister branch of Chloromyxum kurisi. In view of the morphologic, morphometric, and molecular differences with the existing species of Chloromyxum, and considering the differences in hosts and geographic locations, the present species is treated as new and the name Chloromyxum argusi n. sp. is proposed.

Background Microsporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult and immunological techniques such as immunofluorescence have proved to be a useful and reliable tool in the diagnosis of human microsporidiosis. For this reason, we have produced and characterized monoclonal antibodies (MAbs) specific for Encephalitozoon intestinalis (the second most frequent microsporidian infecting humans), and other Encephalitozoon species, that can be used in different diagnostic techniques. Results Seven MAbs were selected in accordance with their optical density (OD). Four (4C4, 2C2, 2E5 and 2H2) were isotype IgG2a; two (3A5 and 3C9) isotype IgG3, and one Mab, 1D7, IgM isotype. The selected monoclonal antibody-secreting hybridomas were characterized by indirect immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), Western blot, immunoelectron microscopy (Immunogold) and in vitro cultures. The study by IFAT showed different behavior depending on the MAbs studied. The MAbs 4C4, 2C2, 2E5 and 2H2 showed reactivity against epitopes in the wall of the spore (exospore and endospore) epitopes located in Encephalitozoon sp. spores, whereas the MAbs 3A5, 1D7 and 3C9 showed reactivity against internal epitopes (cytoplasmic contents or sporoplasm) of E. intestinalis spores. All MAbs recognized the developing parasites in the in vitro cultures of E. intestinalis. Additionally, 59 formalin-fixed stool samples that had been previously analyzed were screened, with 26 (44%) presenting microsporidian spores (18 samples with E. intestinalis and 8 samples with Enterocytozoon bieneusi). Detection of microsporidian spores by microscopy was performed using Calcofluor stain, Modified Trichrome, Quick-Hot Gram Chromotrope, as well as IFAT using MAbs 4C4, 2C2, 2E5 and 2H2. The 4 MAbs tested clearly recognized the larger spores corresponding to E. intestinalis, but showed no reactivity with Enterocytozoon bieneusi spores. The mass spectrometry and proteomic study revealed that the Mabs 4C4, 2C2, 2E5 and 2H2 recognized the Spore Wall Protein 1 (SWP1) as the antigenic target. Conclusions The IFAT-positive MAbs exhibited excellent reactivity against spores and developmental stages, permitting their use in human and animal diagnosis. The epitopes recognized (exospore, endospore and cytoplasmic contents) by the different MAbs developed need further study, and may reveal potential targets for vaccine development, immunotherapy and chemotherapy.

Myxosporean Myxobolus saladensis sp. nov. in the gills ofMugil liza Valenciennes, 1836 from Samborombón Bay was described by light and electron microscopy studies. Spores were pyriform and binucleated, measuring 10.63±0.36 pm (n=20) long, 9.24±0.50 pm (n=20) wide and 4.13±0.36 pm (n=20) thick, included in polysporic cyst-like plasmodia. Elongated pyriform polar capsules were of equal size (3.84±0.27 pm long and 2.30±0.12 pm wide). The sporoplasm contained some sporoplasmosomes. Each PC contained a polar filament with 4-5 coils obliquely arranged in relation to the polar capsules axis. The PC wall was composed of two layers of different electron densities. Based on the morphological and ultrastructure differences of the spore to those of previously described species of Myxobolus, we describe a new species, Myxobolus saladensis sp. nov., Facultad de Ciencias Naturales y Museo

This paper describes light and ultrastructural observations and molecular analysis of a fish-infecting myxosporean, Henneguya gilbert n. sp., which was found infecting the gill epithelium of the commercially important freshwater teleost fish Cyphocharax gilbert (Curimatidae) collected in the estuarine region of Guandu River, Rio de Janeiro State, Brazil. The parasite occurs in the gills, forming whitish spherical to ellipsoidal polysporic cysts measuring up to ~ 750 μm, and displaying asynchronous development. Mature myxospores are ellipsoidal with a bifurcated caudal process. The length, width and thickness of the body of the myxospore are 12.0 × 5.3 × 3.6 μm, respectively; two equal caudal processes are 16.8 μm long, and the total length of the myxospore is 27.2 μm. There are two unequal polar capsules: the larger measures 5.5 μm length × 1.3 μm width and has a polar filament with 9–10 coils; the smaller is 4.0 μm long × 1.3 μm wide and has a polar filament with 7–8 coils. The sporoplasm is binucleated and presents a spherical vacuole surrounded by numerous globular sporoplasmosomes. Phylogenetic analysis, based on the small subunit rRNA sequencing, using maximum likelihood method reveals the parasite clustering together with other myxobolids that are histozoic and parasitize freshwater fish of the order Characiformes, thereby strengthening the contention that the host phylogenetic relationships and aquatic environment are the strongest evolutionary signals for myxosporeans of the family Myxobolidae.

A new species of Myxosporea, Henneguya loreotoensis n. sp. is described parasitizing the gill filaments from 17 of 35 specimens (48.5%) of Corydoras leucomelas (Siluriformes: Callichthyidae) caught in the Nanay River, near village Ninarumi, in the Loreto state, Peru. Mature spores were ellipsoidal in shape from the frontal view, measuring 36.2±0.1μm (36.1-36.3) in total length, 14.3±0.1μm (14.2-14.4) in body length, 5.1±0.1μm (4.9-5.3) in width and 21.9±0.1μm (21.8-22.0) in the caudal process. The two polar capsules were symmetrical and elongated, measuring 5.1±0.1μm (4.9-5.3) in length and 2.4±0.2μm (2.1-2.7) in width, containing a polar filament with five coils arranged obliquely to the longitudinal axis. The sporoplasm was binucleate. Partial sequencing of the ssu-rDNA of H. loretoensis n. sp. resulted in a total of 1676 nucleotides, and this sequence did not match any of the myxozoan available in the GenBank. The phylogenetic analysis shows H. loretoensis n. sp. as a sister species of Henneguya paraensis, another amazonian myxozoan parasite of Cichla temensis (Perciformes: Cichlidae).

During the investigation of actinosporean fauna diversity from commercial fish ponds in Hubei Province, China, a novel aurantiactinomyxon type was found from Branchiura sowerbyi. Spore body of the aurantiactinomyxon was ellipsoidal in side view and triangular in apical view, 15.5 ± 0.5 (14.5–16.4) μm in diameter; three leaf-like caudal processes were approximately equal, measuring 13.2 ± 0.9 (11.5–16.2) μm long and 7.4 ± 0.4 (6.7–8.0) μm wide at the base; three polar capsules were located at the apex of spore body, globular in apical view, 2.2 ± 0.1 (2.0–2.3) μm in diameter, and pyriform in side view, 2.5 ± 0.2 (2.3–2.9) μm in length and 2.0 ± 0.2 (1.8–2.4) μm in width; a total of 32 germ cells were observed within the sporoplasm. Ultrastructural analysis showed that the development was asynchronous between pansporocysts but synchronous within a pansporocyst. The formation of sporoblast and the development of sporogonic stage were also described and discussed. The 18S ribosomal DNA sequences of the current aurantiactinomyxon type corresponded to that of a previously reported Thelohanellus testudineus, suggesting that the newly identified aurantiactinomyxon type is the actinosporean stage in the life cycle of T. testudineus.

Microscopic and molecular procedures are used to describe a new myxosporean species, Henneguya carolina sp. nov., found infecting the intestine of the marine teleost fish Trachinotus carolinus on the southern Atlantic coast of Brazil. Spherical to ellipsoid cysts, measuring up to ~750 µm, display synchronous development. Mature myxospores are ellipsoidal with a bifurcated caudal process. Myxospore body length, width, and thickness are 12.7 ± 0.8 (12.0-13.4) µm, 8.8 ± 0.6 (7.5-9.6) µm, and 5.8 ± 0.4 (5.0-6.4) µm, respectively; 2 equal caudal processes are 16.8 ± 1.1 (15.9-18.0) µm long, and the total myxospore length is 29.4 ± 0.8 (28.4-30.4) µm. Two pyriform polar capsules measure 5.0 ± 0.5 (4.6-5.6) × 2.4 ± 0.4 (1.9-2.9) µm, and each contains a polar filament forming 3 to 4 coils. Sporoplasm is binucleated and presents a spherical vacuole surrounded by numerous globular sporoplasmosomes. Molecular analysis of the small subunit rRNA gene by maximum parsimony, neighbor joining, and maximum likelihood reveals the parasite clustering together with other myxobolids that are histozoic in marine fish of the order Perciformes, thereby strengthening the contention that the host phylogenetic relationships and aquatic environment are the strongest evolutionary signal for myxosporeans of the family Myxobolidae.

Mucus-derived nucleosides serve as key host cues for myxozoan actinospore fish host recognition, but to date their use for experimental actinospore activation in the laboratory or application in disease prevention has not progressed very far. One obstacle has been the low solubility of pure inosine and guanosine. To overcome this, we used inosine-arginine salt (ino-arg), which incorporates both high activation properties and high solubility. We tested its efficacy both in microassays directly observing reactions of actinospores of 2 distantly related myxozoan species, Myxobolus cerebralis and M. pseudodispar in comparison to inosine, as well as its actinospore-inactivation properties by premature polar capsule discharge in an infection experiment. Ino-arg was considerably more effective in eliciting polar capsule discharge and sporoplasm emission at much lower concentrations than pure inosine and, in contrast to the latter, remained dissolved in aqueous solution. Ino-arg exposure of M. pseudodispar actinospores resulted in polar capsule discharge and sporoplasm emission before host contact and subsequently in a lower infection rate in roach Rutilus rutilus.

Henneguya jocu n. sp. (Myxosporea, Myxobolidae) is described from the gill lamellae of the marine teleost fish Lutjanus jocu, with a focus on ultrastructural and molecular features. This myxosporean forms subspherical cysts up to ∼260 μm × 130 μm long, and develops asynchronously. Mature myxospores ellipsoidal with a bifurcated caudal process. Myxospore length 10.9 ± 0.4 μm (n = 50); width, 8.2 ± 0.3 μm (n = 50); and thickness, 2.9 ± 0.5 μm (n = 50). Two equal caudal processes, 34.1 ± 1.0 μm long (n = 50); and total myxospore length, 45.2 ± 1.0 μm (n = 50). Two symmetric valves surround two ellipsoidal polar capsules, 5.0 ± 0.3 × 1.4 ± 0.2 μm (n = 20), each containing an isofilar polar filament forming 4–5 coils along the inner wall of these structures, as well as a binucleated sporoplasm presenting a spherical vacuole and several globular sporoplasmosomes. Both the morphological data and molecular analysis of the SSU rDNA gene identify this parasite as a new species of the genus Henneguya. Maximum Likelihood and Maximum Parsimony analyses further indicate that the parasite clusters within others marine Myxobolidae species, forming a group alongside other Henneguya species described from marine hosts.

Microsporidian spores contain a single polar filament that is coiled around the interior of the spore. Upon germination the polar tube (post-germination polar filament) is ejected by inversion into a host cell. The sporoplasm flows through the polar tube, directly infecting the cytoplasm of the cell. Various species of microsporidia display differences in the number of coils in the polar filament and in the amino acid sequence of the polar tube proteins (PTPs). Nosema pernyi is a lethal pathogen that causes microsporidiosis in the Chinese oak silkworm, Antheraea pernyi. In this study, we identified three PTPs in N. pernyi using RT-PCR and LC-MS/MS. Polar tube protein 3 was localized in the polar tube using immuno-histochemical staining and an immunofluorescence assay. Co-immunoprecipitation data and LC-MS/MS analysis revealed that some potential proteins, like immune related proteins in A. pernyi may interact with PTP3.

Made available in DSpace on 2019-10-06T16:35:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-01 Plasmodia containing spores of a new species of myxozoan, Henneguya jundiai n. sp., were found in the gill arches of Rhamdia quelen catfish. The present study describes H. jundiai n. sp. based on morphological and molecular analysis. The mature spores were elongated and measured 26.9 ± 1.9 (22.9–29.2) μm in total spore length, 9.5 ± 0.4 (8.8–10.0) μm in spore body length, 4.6 ± 0.4 (4.1–5.5) μm in spore body width, 17.3 ± 1.8 (14.1–19.8) μm in tail length, 4.9 ± 0.3 (4.6–5.5) μm in polar capsule length and 1.4 ± 0.2 (1.2–1.7) μm in polar capsule width. The polar filaments had 6–7 coils. Histological analysis revealed the formation of a large plasmodium connected to the gill arch through the epithelial tissue. Ultrastructural analysis revealed the presence of a binucleated sporoplasm, as well as the polar capsules and the coils of the polar filament. Phylogenetic analysis found that H. jundiai n. sp. is located within a subclade formed by species of Henneguya which parasitize siluriform fish from Brazil. Henneguya jundiai n. sp. appears as a sister species of H. quelen. The results of the analyzes clearly indicated the existence of a new species, H. jundiai. Departament of Parasitology Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, Botucatu Docente do Programa de Pós-graduação em Análise de Sistemas Ambientais Centro Universitário CESMAC Departament of Parasitology Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, Botucatu

Two new myxosporean species, Myxobolus marumotoi n. sp. and Cardimyxobolus japonensis n. sp. (Myxozoa: Myxosporea: Bivalvulida), are described from the dark sleeper, Odontobutis obscura, in Japan, based on their morphological and molecular characterizations. Plasmodia of M. marumotoi n. sp. (Myxobolidae) grew in the myofiber of trunk muscles, forming pseudocysts. The rounded spore was relatively large, measuring 13.3-15.0 μm (average 13.8) in length, 14.2-15.0 μm (14.6) in width, and 10.0-11.7 μm (10.8) in thickness, with two subspherical polar capsules of 7.9-9.6 μm (8.4) in length by 5.4-6.3 μm (5.9) in width (n = 15). The polar capsules were directed toward the apex of the spore, packing five to six spirals of the polar filament. Plasmodia of C. japonensis n. sp. (Ortholineidae) were surrounded by thin fibrous tissue, forming cysts in the lamina propria of the alimentary tract. The spore was ovoid, wider than long, in valvular view and spindle-shaped in sutural view. It measured 8.8-10.4 μm (9.4) in length, 11.3-12.5 μm (11.9) in width, and 5.2-6.7 μm (5.8) in thickness, with two ovoid polar capsules of 4.2-5.0 μm (4.7) in length by 2.9-3.8 μm (3.3) in width (n = 15). The shell valves of spores often showed a flattened anterior border and semicircular posterior border, and the two polar capsules were directed toward opposite lateral sides. In addition, the sporoplasm contained an iodinophilous vacuole. Almost complete small-subunit (SSU) rDNA sequences, except for primer flanking regions, were obtained for both species; 1,996 bp long for the former and 1,588 bp long for the latter. On phylogenetic trees based on the SSU rDNA sequences of representative species of Bivalvulida, M. marumotoi n. sp. and C. japonensis n. sp. formed a distinct branch in the Henneguya/Myxobolus clade or near but outside this clade, respectively. This study is the first report of the genetic characterization for the genus Cardimyxobolus.

A new myxosporean, Zschokkella auratis sp. nov., infecting the gall bladder of the gilthead seabream Sparus aurata in a southern Portuguese fish farm, is described using micro- scopic and molecular procedures. Plasmodia and mature spores were observed floating free in the bile. Plasmodia, containing immature and mature spores, were characterized by the formation of branched glycostyles, apparently due to the release of segregated material contained within numerous cytoplasmic vesicles. Mature spores were ellipsoidal in sutural view and slightly semicir- cular in valvular view, with rounded ends, measuring 9.5 ± 0.3 SD (8.7�10.3) µm in length and 7.1 ± 0.4 (6.5�8.0) µm in width/thickness. The spore wall was composed of 2 symmetrical valves united along a slightly curved suture line, each displaying 10 to 11 elevated surface ridges. Two equal sub- spherical polar capsules, 3.7 ± 0.3 (3.0�4.1) µm long and 3.0 ± 0.2 (2.6�3.2) µm wide, were located separately at the spore's extremities. Each polar capsule contained a polar filament forming 4 to 5 coils. The sporoplasm was binucleate and contained numerous sporoplasmosomes. Morphological data, tissue tropism, and molecular analysis of the small subunit rDNA gene identified this parasite as a new species of Zschokkella. Maximum parsimony, neighbor-joining, and maximum likelihood inferences clustered the parasite in a subclade containing other Zschokkella species parasitizing the gall bladder of brackish and marine fish hosts, located within the coelozoic clade of the major freshwater clade; this supports the existence of a marine subclade within the 'freshwater' clade, as well as the existence of a correlation between tissue tropism and myxosporean phylogeny.

Myxosporeans (Myxozoa) are eukaryotic parasites, primarily of fish, whose classification is in a state of flux as taxonomists attempt to synthesize the traditional morphology-based system with emerging DNA sequence-based phylogenies. The genus Sphaerospora Thelohan, 1892, which includes pathogenic species that cause significant impacts on fisheries and aquaculture, is one of the most polyphyletic taxa and exemplifies the current challenges facing myxozoan taxonomists. The type species, S. elegans, clusters within the Sphaerospora sensu stricto clade, members of which share similar tissue tropism and long insertions in their variable rRNA gene regions. However, other morphologically similar sphaerosporids lie in different branches of myxozoan phylogenetic trees. Herein, we significantly extend taxonomic sampling of sphaerosporids with SSU + LSU rDNA and EF-2 sequence data for 12 taxa including three representatives of the morphologically similar genus Polysporoplasma Sitja-Bobadilla et Alvarez-Pellitero, 1995. These taxa were sampled from different vertebrate host groups, biogeographic realms and environments. Our phylogenetic analyses and statistical tests of single and concatenated datasets revealed Sphaerospora s. s. as a strongly supported monophyletic lineage, that clustered sister to the whole myxosporean clade (freshwater + marine lineages). Generally, Sphaerospora s. s. rDNA sequences (up to 3.7 kb) are the longest of all myxozoans and indeed metazoans. The sphaerosporid clade has two lineages, which have specific morphological, biological and sequence traits. Lineage A taxa (marine Sphaerospora spp.) have a single binucleate sporoplasm and shorter AT-rich rDNA inserts. Lineage B taxa (freshwater/brackish Sphaerospora spp. + marine/brackish Polysporoplasma spp.) have 2–12 uninucleate sporoplasms and longer GC-rich rDNA inserts. Lineage B has four subclades that correlate with host group and habitat; all Polysporoplasma species, including the type species, cluster together in one of these subclades. We thus suppress the genus Polysporoplasma and the family Polysporoplasmidae and emend the generic diagnosis of the genus Sphaerospora. The combination of morphological, biological and DNA sequence data applied in this study helped to elucidate an important part of the taxonomic puzzle within the phylum Myxozoa.

Kudoa septempunctata is a myxosporean parasite of Paralichthys olivaceus (olive flounder) and causes a foodborne illness that affects more than 100 cases in Japan each year. We previously reported that the consumption of raw olive flounder meat containing a high concentration of K. septempunctata spores induces transient but severe diarrhea and emesis through an unknown mechanism. Here, we demonstrate that K. septempunctata sporoplasm plays an important role in mediating the toxicity of K. septempunctata. When K. septempunctata spores were inoculated in Caco-2 human intestinal cells, K. septempunctata sporoplasms were released from spores, and they invaded the cells. Electron microscopic observations revealed that the sporoplasm invasion severely damaged the Caco-2 cells. The inoculation of K. septempunctata spores eliminated the transepithelial electrical resistance (TER) across the cell monolayer. Inhibiting the invasion of the sporoplasms prevented the observed loss in cell layer integrity, as illustrated by the rapid elimination of the TER. These results suggest that the invasion by sporoplasms severely damaged individual intestinal cells, resulting in a loss of cell monolayer integrity.

Pleistophora macrozoarcidis a microsporidian parasite infecting the muscle tissue of the ocean pout Macrozoarces americanus collected from the Gulf of Maine of the Atlantic Ocean, MA, USA, was morphologically described on the basis of ultrastructural features. Infection was detected as opaque white or rusty brown lesions scattered throughout the musculature of the fish mainly in the region anterior to anus. Transmission electron microscopy showed that in individual parasitized muscle cells, the infection progresses within parasite formed vesicles which are in direct contact with muscle cell elements. The earliest observed parasitic stages are the globular multinucleated proliferative cells or plasmodia limited by a highly tortuous plasmalemma with intervesicular finger-like digitations projecting into the parasite cytoplasm. These cells divided through the invagination of the plasmalemma and the amorphous coat producing daughter-cells. Fine electron-dense secretion is deposited on the plasmalemma that causes its thickening which is a sign of commencement of the sporogonic phase. This phase is carried out by cytokinesis of the sporonts and results in the formation of sporoblasts and finally spores. Mature spore has a thin electron-dense exospore, a thick electron-lucent endospore, and the plasma membrane which encloses the spore contents. A single nucleus is centrally located with the posterior region containing a posterior vacuole. The majority of spores have 7–13 coils in 1–2 rows, and a small group of spores had about 23 coils forming two rows. Events of polar filament extrusion for penetration of uninfected cells were studied. The polaroplast membranes were expanded and occupy most of the length of the spore. The coils are dislocated from the sides of the spore to throughout the entire sporoplasm. The polar filament everts and extrudes through the polar cap with a sufficient force to pierce adjacent sporophorous vesicle walls. After eversion, the polar filament is referred to as a polar tubule, as it forms a tube through which the sporoplasm travels. It pierces anything in its path and deposits the sporoplasm at a new location to begin another infective cycle.

Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens., Author summary Microsporidia are obligate intracellular parasites that cause disease in immune suppressed individuals such as those with HIV/AIDS and recipients of organ transplants. The microsporidia are defined by a unique invasion organelle, the polar tube. The formation of this organelle and its role in the mechanism of infection remain unknown. Herein, we have identified a role for Encephalitozoon hellem polar tube protein 4 (PTP4) in infection demonstrating that PTP4 can bind to the host cell surface via the host transferrin receptor 1 (TfR1) protein. Interfering with the interaction of PTP4 and TfR1 causes a significant decrease in microsporidian infection of host cells. These data suggest that PTP4 functions as an important microsporidian protein during host cell infection by this pathogen.

Cryopreservation methods for Kudoa septempunctata have not been established. This prevents an effective study of K. septempunctata, which cannot be artificially cultivated in the laboratory. In this study, we attempted to establish a cryopreservation method for K. septempunctata sporoplasm using Cellbanker® 1, a commercial preservation medium for mammalian cells. Spores were purified from the meat of Paralichthys olivaceus (olive flounder). These purified spores were suspended in Cellbanker® 1 and were stored at −80 °C for up to 16 months. Although the spores stored at −80 °C for 16 months were damaged, the sporoplasms maintained its amoeba-like indeterminate morphology, and their motility was well preserved. The viability of sporoplasms was variable among vials but was not below 70 %. In addition, the sporoplasms stored at −80 °C for 16 months could decrease the transepithelial electrical resistance of Caco-2 cells. These results indicate that this cryopreservation method using Cellbanker® 1 could preserve the viability and pathogenesis of K. septempunctata sporoplasm.

Actinosporean infection of oligochaetes living in the mud of a commercial gibel carp pond with myxosporean disease was studied. Six actinospore types were detected exclusively from the oligochaete Branchiura sowerbyi Beddard with very high prevalence (18%). Five out of the six types were identified as the same actinosporeans described in previous reports, the sixth actinosporean was identified as a new Neoactinomyxum type and described here based on morphological and molecular characterisation. Spore body of the actinospore was globular, much smaller than caudal processes. Three caudal processes were disc-like in apical view, hemispherical in side view, closer together and encircling the spore body. The number of sporoplasm cells was detected as eight in one specimen. The new actinosporean markedly differed from other Neoactinomyxum types in literature having much bigger caudal processes. DNA sequence analyses further confirmed the morphological identification, and revealed the actinosporean described here (KU641392) possessed less than 94% sequence similarity with myxozoans available in the GenBank database.

The ultrastructure of the spores and developmental stages of Ellipsomyxa mugilis in Nereis diversicolor were studied by transmission electron microscopy. The ultrastructure features and the developmental stages show many similarities with the general pattern described for other actinospores. However, several new features are definitely worth noting. For example, tetranucleated cells precede the formation of the initial pansporocyst, which preserves the 2 original enveloping cells until the end of sporogony. In the initial stages of sporogony, the future sporoplasm cell acquires the first secondary cell by an engulfment process. In the final stage of sporogony, spores are formed by a sporoplasm with 2 secondary cells and 1 somatic nucleus, and the polar capsule has a polar filament with a helicoidal arrangement possessing 7–8 coils.

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.

This study identifies and characterizes one novel type of triactinospomyxon in oligochaete specimen of Branchiura sowerbyi Beddard collected from a fish pond used for rearing gibel carp located in Caidian Experimental Station of the Institute of Hydrobiology. It is nominated as Triactinospomyxon caidianensis type. The spore is of characteristic triactinomyxon "anchor" shape and possesses a spore body with sporoplasm containing 32 germ cells, 3 polar capsules, and 3 caudal processes. Compared with other triactinomyxon spores described previously, T. caidianensis type has a short spore axis with 76.5 mu m in length and a very short style with 38.9 mu m in length. Molecular analyses on 18S rDNA sequences indicate that the novel T. caidianensis type is most closely related to Triactinomyxon sp SA-2005, Antonactinomyxon sp KAB-2001, and Synactinomyxon sp KAB-2001 with 80.33% to 81.92% identities. On the basis of spore morphology and genetic data, the T. caidianensis type presented in this paper differs from those already known and described in the literatures.