Polar Tube Research Articles

Human Microsporidiosis: A Narrative Review of Serodiagnostic Tests

Context: Microsporidiosis is an emerging disease in both immunocompetent and immunocompromised patients. Diagnosis of human microsporidiosis is challenging. Parasitological tests using staining methods have limitations for detecting spores in stool samples. The performance of molecular methods for diagnosing human microsporidiosis is satisfactory, but these methods are expensive and time-consuming. Serodiagnosis tests for the detection of antibodies have insufficient specificity. Detecting microsporidia antigens seems to be an appropriate method for diagnosing human microsporidiosis. Therefore, the present study reviewed serological approaches to assess the current status of these methods for diagnosing human microsporidiosis. Evidence Acquisition: All related published articles were searched. The search terms were “microsporidiosis”, “microsporidiasis”, “human microsporidiosis”, and “human microsporidiasis”, combined with the search terms “diagnosis”, “serodiagnosis”, “immunodiagnosis”, “antigen detection”, and “antibody detection”. Data were extracted from the articles that met our eligibility criteria. Immunodiagnosis studies in experimentally infected animals were excluded. Results: Few studies have evaluated the performance of serological tests for diagnosing human microsporidiosis. The IgG response against spore wall protein or polar tube antigens remains at least three years after infection and shows more cross-reactivity with other species. Therefore, the western blot should be conducted as a confirmatory method. Serodiagnosis tests using monoclonal antibodies against microsporidia showed less cross-reactivity and more efficacy compared to polyclonal antibodies. Conclusions: Indirect fluorescent antibody test based on the monoclonal antibody is highly effective in diagnosing microsporidia species in clinical samples.

Host-Parasite Interaction and Phylogenetic of a New Cnidarian Myxosporean (Endocnidozoa: Myxobolidae) Infecting a Valuative Commercialized Ornamental Fish from Pantanal Wetland Biome, Brazil.

Myxozoans are a diverse group of parasitic cnidarians of wide distribution. A new species, Myxobolus matogrossoensis n. sp., is herein described infecting wild specimens of tetra mato-grosso Hyphessobrycon eques, caught in the Pantanal biome, the world’s largest tropical wetland area. Cysts were found in 3 of the 30 examined fishes. Mature myxospores were ovoid in shape in frontal and measured 6.6 ± 0.4 µm (6.2–7.0 µm) in length and 3.5 ± 0.2 µm (3.3–3.7 µm) in width. The two polar capsules were elongated in shape, equal in size and occupying almost half of the myxospore body. They measured 3.3 ± 0.2 µm (3.1–3.5 µm) in length and 1.8 ± 0.1 µm (1.7–1.9 µm) in width. The polar tubules presented three to four turns. Phylogenetic analysis placed the new species within a clade containing myxobolid species from South American characiforms fish and appears as a close species of Myxobolus piraputangae and Myxobolus umidus. Nevertheless, the sequences of the new species and P. umidus and P. piraputangae have a large genetic divergence of 12 and 12.2% in their 18S rDNA gene, respectively. To the best of our knowledge, this is the first report of a Myxobolus species parasitizing the tetra fish mato-grosso, thus increasing our knowledge of cnidarian myxosporean diversity from South America.

Morphological and Molecular Characterization of Myxobolus nkondjockei sp. nov. (Myxozoa: Myxobolidae), A Parasite of Labeo batesii Boulenger, 1911 (Teleostei: Cyprinidae) from Makombè River in Cameroon.

Myxozoans are economically important group of metazoan parasites, which can cause diseases in a large variety of commercially important fishes. Increased knowledge on molecular features has shown that traditional descriptive characters may be misleading. Combination of both descriptive and molecular features is therefore necessary for an integrated taxonomic assessment. Cyprinid Labeo batesii, sampled in the Makombè River at Nkondjock in Cameroon were examined for myxosporeans. Identification of parasite species was based on morphological and molecular sequence analyses of myxospores. Phylogenetic analysis was performed using maximum likelihood (ML) and Bayesian inference (BI) methods. The scales of L. batesii were infected by Myxobolus nkondjockei sp. nov Their mature myxospores are ovoid in frontal view and lenticular in lateral view, with two rounded ends. These myxospores measured 10.3 (10-10.9) μm length and 8.0 (7.3-8.5) μm width. Myxospores have two ovoid and equal sizes polar capsules. They measured 4.5 (4.0-5.0) µm in length and 2.4 (2-2.9) µm in width. Polar tubules were coiled in 4-5 turns perpendicular to the longitudinal axis of the polar capsules. Phylogenetic analysis of the 18S rDNA sequence show clustering of M. nkondjockei sp. n. close to an undetermined species Myxobolus sp. reported infecting gill lamellas of Labeo rohita from India. The morphological, molecular and phylogenetic data provided for M. nkondjockei sp. n. are solid basis for further identification of this myxozoan of which pathogenicity probably plays an economic role at culturing the hosts.

Maturation of subtilisin-like protease NbSLP1 from microsporidia Nosema bombycis.

Microsporidia are obligate intracellular parasites and possess a unique way of invading hosts, namely germination. Microsporidia are able to infect almost all animal cells by germination. During the process, the polar tube extrudes from the spores within, thus injecting infectious sporoplasm into the host cells. Previous studies indicated that subtilisin-like protease 1 (NbSLP1) of microsporidia Nosema bombycis were located at the polar cap of germinated spores where the polar tube extrusion. We hypothesized that NbSLP1 is an essential player in the germination process. Normally, SLP need to be activated by autoproteolysis under conditions. In this study, we found that the signal peptide of NbSLP1 affected the activation of protease, two self-cleavage sites were involved in NbSLP1 maturation between Ala104Asp105 and Ala124Asp125 respectively. Mutants at catalytic triad of NbSLP1 confirmed the decreasing of autoproteolysis. This study demonstrates that intramolecular proteolysis is required for NbSLP1 maturation. The protease undergoes a series of sequential N-terminal cleavage events to generate the mature enzyme. Like other subtilisin-like enzymes, catalytic triad of NbSLP1 are significant for the self-activation of NbSLP1. In conclusion, clarifying the maturation of NbSLP1 will be valuable for understanding the polar tube ejection mechanism of germination.

Antifungal activity of “HO21-F”, a formulation based on Olea europaea plant extract, in honey bees infected with Nosema ceranae

Nosema ceranae is a microsporidium parasite that silently affects honey bees, causing a disease called nosemosis. This parasite produces resistant spores and germinates in the midgut of honey bees, extrudes a polar tubule that injects an infective sporoplasm in the host cell epithelium, proliferates, and produces intestinal disorders that shorten honey bee lifespan. The rapid extension of this disease has been reported to be widespread among adult bees, and treatments are less effective and counterproductive weakening colonies. This work aimed to evaluate the antifungal activity of a prototype formulation based on a non-toxic plant extract (HO21-F) against N. ceranae. In laboratory, honey bees were infected artificially, kept in cages for 17 days and samples were taken at 7 and 14 days post infection (dpi). At the same time, in field conditions we evaluated the therapeutic effect of HO21-F for 28 days in naturally infected colonies. The effectiveness of the treatment has been demonstrated by a reduction of 83.6 % of the infection levels observed in laboratory conditions at concentrations of 0.5 and 1 g/L without affecting the survival rate. Besides, in-field conditions we reported a reduction of 88 % of the infection level at a concentration of 2.5 g/L, obtaining better antifungal effectiveness in comparison to other commercially available treatments.As a result, we observed that the use of HO21-F led to an increase in population size and honey production, both parameters associated with colony strength. The reported antifungal activity of HO21-F against N. ceranae, with a significant control of spore proliferation in worker bees, suggests the promising commercial application use of this product against nosemosis, and it will encourage new research studies to understand the mechanism of action, whether related to the spore-inhibition effect and/or a stimulating effect in natural response of colonies to counteract the disease.

Sphaeromyxa cornuti n. sp., a New Species of Myxosporean Infecting the Gallbladder of the Moorish Idol, Zanclus cornutus (Linnaeus, 1758) from Lakshadweep Waters.

The present study describes a new species of myxosporean, Sphaeromyxa cornuti n. sp. infecting the gallbladder of the Moorish idol, Zanclus cornutus (Linnaeus 1758) collected from Lakshadweep waters of the Arabian Sea. Fish were collected using traps and cages. The morphology of mature spores recovered from the gallbladder of Z. cornutus was studied under Nomarski Differential Interference Contrast (DIC) optics. The molecular and phylogenetic analyses were based on SSU rDNA. Sphaeromyxa cornuti n. sp. is characterized by arcuate myxospores with tapering extremities and round ends in valvular, and slightly sigmoid in sutural views (19.2-24.7 µm × 4.1-5.7µm). The two polar capsules are unequally elongate-ovoid in shape and positioned at opposite ends of the spore (6.2-9.7µm × 1.7-2.6µm). Each encloses an irregularly folded, ribbon-like polar tubule, which is oriented parallel to polar capsule axis. In molecular and phylogenetic analyses, the present myxosporean revealed significant differences with related forms and clustered together with S. hellandi within the 'incurvata' group of the Sphaeromyxa clade with high nodal support. Morphological, morphometric, molecular and phylogenetic differences between our material and previously described species of Sphaeromyxa, along with host and geographic variations indicate that the present myxosporean is unique and the name Sphaeromyxa cornuti n. sp. is proposed. This forms the first report of a myxosporean parasite-infecting Z. cornutus.

A new microsporidian parasite, Microsporidium theragrae n. sp., infecting Alaska pollock Gadus chalcogrammus (Teleostei: Gadidae).

A new microsporidian infecting Gadus chalcogrammus Pallas, 1814 (Gadidae), is described based on morphological, ultrastructural, and molecular studies. This microsporidian parasite develops inside intramuscular spindle-shaped lesions measuring approximately 1-2mm in width and 4-8mm in length. Infected cells encapsulated by a host-produced wall containing a sponge-like acellular zone. Sporogony presumably proceeds via segmentation of sporogonial plasmodium, resulting in a variable number of spores. Sporogonial stages develop in sporophorous vesicles (SVs), abutting a moderately electron-dense thick walled coat of a homogeneous amorphous material. SVs space contains rare granular and tubular inclusions. Neighboring SVs often interconnected by bridges of the host cell cytoplasm that were limited by membrane comparable with SV coat. The elongate-ovoid spores, measuring 4.29 ± 0.38 × 2.51 ± 0.26μm (N 104), possess a bipartite polaroplast and polar tube with 15-16 coils arranged in 2-3 layers. The angle of tilt of the polar tube coils is less than 30°. The sequence analysis of SSU rDNA coding region showed that the studied microsporidians differs from other fish muscle-infecting species at least in 17bp (2.58%) and is closely related to Microsporidium cypselurus Yokoyama et al. (2002) infecting the flying fish from East China Sea. The parasite is provisionally positioned as Microsporidium theragrae sp. n.

Morphological and molecular characterization of the cryptic species Myxobolus cataractae n. sp. (Cnidaria: Myxozoa: Myxobolidae) parasitizing Imparfinis mirini (Siluriformes: Heptapteridae).

We describe Myxobolus cataractae n. sp., found parasitizing Imparfinis mirini from Cachoeirinha stream, a tributary of Pardo River, Paraná River basin, municipality of Botucatu, São Paulo State, Brazil. We based the description on myxospore morphology, histology, and small subunit ribosomal DNA partial sequences. Whitish and elongated mass of myxospores were found in gill filaments, occupying an intrafilamental position. The myxospores were slightly ellipsoidal and the morphometric measurements revealed a myxospore body length of 7.8±0.4μm; myxospore width of 5.9±0.4μm; and thickness of 3.9±0.3μm. The polar capsules are equal in size and measured 3.5±0.2μm in length and 1.7±0.2μm in width. The polar tubules had 6-7 coils. Myxobolus cataractae n. sp. is classified as the intrafilamental type and chondreal subtype by the histological analysis of gills. Phylogenetic analysis showed a well-supported subclade formed by species that parasitize gills of Siluriformes, with M. imparfinis as a sister species of Myxobolus cataractae n. sp. Using molecular and morphological characterization, this cryptic species was identified as a new species of the genus Myxobolus.

Proteomic profile of polar filament and polar tube from fungal pathogen microsporidium Nosema bombycis provides new insights into its unique invasion organelle

Microsporidium is a kind of intracellular fungal pathogen that greatly threatens the human health, breeding industry, and food security. All members of microsporidia possess a unique, highly specialized invasion organelle, described as the polar filament. Like “reversing a finger of gloves”, the polar filament discharges out of mature spores to transform as the polar tube, and pathogenic sporoplasm is transported to host cell through polar tube to complete infection. During the invasion process, the structure of polar filament and polar tube has changed, so does the protein composition on them? In this study, we firstly proposed a purification method for polar filament and polar tube from microsporidium Nosema bombycis which was infected silkworm Bombyx mori, and it was also found that the structure of polar filament and polar tube was obviously different. Therefore, the proteome of these two structures was comparatively analyzed. A total of 881 and 1216 proteins were respectively identified from the polar filament and polar tube. Ten potential novel polar tube proteins (PTPs) were screened, providing a reference for the novel PTPs identification. Compared with the polar filament, there were 35 upregulated and 41 downregulated proteins on the polar tube. GO and KEGG pathway analysis of all proteins from the polar filament and polar tube provided us with a profound understanding for the microsporidian germination process, which was of great significance for clarifying the infection mechanism of microsporidia. SignificanceMicrosporidia are obligate intracellular parasites that infect a wide variety of hosts, including humans. The polar filament is a unique invasion organelle for microsporidia, and it is also one of the important indexes of microsporidian taxonomy. The polar tube is deformed from the primitive polar filament in mature spores. During the germination, the polar filament turns into a polar tube, like “reversing a finger of gloves”, through which pathogenic sporoplasm is transported to host cells to complete infection. Since the structure of the polar filament and polar tube has changed, what about their protein composition? In this study, it was the first time to purify the polar filament and the polar tube from microsporidium Nosema bombycis that was infected silkworm Bombyx mori, which provided new insights for studying the invasion organelle of microsporidia. Comparing the fine structure of polar filament and polar tube, we found that their structure was obviously different. Therefore, the protein composition of these two structures is supposed to be varied. In this case, the proteome of these two structures was comparatively analyzed. A total of 881 and 1216 proteins were respectively identified from the polar filament and polar tube. Ten potential novel polar tube proteins (PTPs) were screened, providing a reference for the novel PTPs identification. Compared with the polar filament, there were 35 upregulated and 41 downregulated proteins on the polar tube. GO and KEGG pathway analysis of all proteins from the polar filament and polar tube provided us with a profound understanding for the microsporidian germination process, which was of great significance for clarifying the infection mechanism of microsporidia.

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.

Morphological and molecular studies on two myxosporean infections of cyprinid fishes: Thelohanellus pyriformis from tench and Thelohanellus cf. fuhrmanni from nase.

During surveys on myxosporean parasites of Lake Balaton and River Danube fishes, two Thelohanellus spp. were found on tench (Tinca tinca) and on common nase (Chondrostoma nasus). They were identified as Thelohanellus pyriformis and Thelohanellus cf. fuhrmanni, respectively. Myxospores of T. pyriformis from tench were collected from arteria branchialis afferens of gill filaments. The mature myxospores of this species were pyriform in shape and 19 ± 0.6l (18–19.5) long, 8.2 ± 0.54 (7.5–9) wide, 7.3 ± 0.25 (7–7.5) thick containing polar tubules with 9–10 turns. The plasmodia of Thelohanellus cf. fuhrmanni were collected from under the skin of snout of the common nase. The myxospores were pyriform, 16.3 ± 0.39l (15.5–16.5) long, 6.5 ± 0.55 (6.3–7) wide, 6.3 ± 0.53 (5.8–7) thick containing polar tubules with 6 turns. Small subunit ribosomal DNA sequences of both Thelohanellus species differed from other known myxozoans. The myxospores morphology, histopathology and ssrDNA sequences supported a diagnosis of T. pyriformis from tench and T. cf. fuhrmanni from common nase.

G lugea sp. infecting Sardinella aurita in Algeria.

Parasitological examination of the commercially important pelagic fish Sardinella aurita Valenciennes, 1847 (Clupeidae) from the Eastern coast of Algeria revealed xenomas in the peritoneal cavity, suggesting a microsporidian infection. The prevalence of the disease was approximately 30% on average, higher in smaller individuals and showing significant seasonal variation. The xenomas contained numerous ellipsoidal spores, surrounded by a dense layer of connective tissue. Spore sizes were 6.10 ± 0.38µm length and 3.54 ± 0.43µm width. Ultrastructural examination by transmission electron microscopy showed various development stages of the parasite, including meronts, sporonts, sporoblasts and mature spores. The internal organization of the mature spores, with a single nucleus, prominent posterior vacuole, a lamellar polaroplast and an isofilar polar tube arranged in a single row, was typical of the genus Glugea. The DNA sequence of the small subunit ribosomal RNA gene confirmed that this parasite belongs to the genus Glugea. Genetic and morphologic comparison with G. sardinellensis, a species previously described in the same host from Tunisia shows many similarities, although some molecular and morphometric inconsistencies precluded the unambiguous assignment of our samples to G. sardinellensis. At the same time, we do not find sufficient grounds to erect a new taxon for our parasite. We discuss the implications of our findings for the current state of the systematics of Glugea.

Waffle Method: A general and flexible approach for improving throughput in FIB-milling

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens with vitrification issues, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and specimens that distribute poorly across grid squares. Here we present a general approach called the ‘Waffle Method’ which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. We propose the Waffle Method as a way to achieve many advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.

Ricin B lectin-like proteins of the microsporidian Encephalitozoon cuniculi and Anncaliia algerae are involved in host-cell invasion.

Microsporidia are obligate intracellular pathogens capable of infecting a wide variety of hosts ranging from invertebrates to vertebrates. The infection process requires a step of prior adherence of Microsporidia to the surface of host cells. A few studies demonstrated the involvement of proteins containing a ricin-B lectin (RBL) domain in parasite infection. In this study Anncalia algerae and Encephalitozoon cuniculi genomes were screened by bioinformatic analysis to identify proteins with an extracellular prediction and possessing RBL-type carbohydrate-binding domains, being both potentially relevant factors contributing to host cell adherence. Three proteins named AaRBLL-1 and AaRBLL-2 from A. algerae and EcRBLL-1 from E. cuniculi, were selected and comparative analysis of sequences suggested their belonging to a multigenic family, with a conserved structural RBL domain despite a significant amino acid sequence divergence. The production of recombinant proteins and antibodies against the three proteins allowed their subcellular localization on the spore wall and/or the polar tube. Adherence inhibition assays based on pre-treatments with recombinant proteins or antibodies highlighted the significant decrease of the proliferation of both E. cuniculi and A. algerae, strongly suggesting that these proteins are involved in the infection process.

Two novel species of Myxobolus parasitizing the gills of Semaprochilodus insignis in the Brazilian Amazon

The present study describes two new Myxobolus species infecting the gills of Semaprochilodus insignis, the most consumed freshwater fish species in the Brazilian Amazon. The fish specimens were caught in the Tapajós River, in the state of Pará, and the morphological, ultrastructural, small subunit ribosomal DNA (ssrDNA), and phylogenetic data of the myxosporean species were obtained. Two species of Myxobolus were found in the gills of S. insignis. Myxobolus maiai n. sp. developed in the gill filaments, and mature myxospores were round-shaped from the frontal view, measuring 12.5–14.8 (13.9 ± 0.5) μm in length, 11.4–13.8 (12.3 ± 0.5) μm in width, and have a thickness of 6.4–7.7 (6.9 ± 0.6) μm in the lateral view, with symmetric values. Its polar capsules were 4.4–6.6 (5.5 ± 0.5) μm in length and 2.3–3.7 (3.0 ± 0.3) μm in width, and the polar tubules had 4 - 5 coils. Myxobolus iarakiensis n. sp. was found infecting the gill arch. Mature myxospores were oval-shaped from the frontal view, and measured 6.7–8.6 (8.0 ± 0.4) μm in length, 4.5–6.3 (5.6 ± 0.4) μm in width, and had a thickness of 2.7–4.7 (3.8 ± 0.5) μm in the lateral view, with symmetric values. Its polar capsules were 2.1–3.7 (2.9 ± 0.3) μm in length and 1.1–2.0 (1.5 ± 0.2) μm in width, and its polar tubules had 4 - 5 coils. The ssrDNA based phylogeny showed these two novel species as grouping in a clade composed of parasite species of Prochilodontidae hosts.

Comparative Transcriptome Investigation of Nosema ceranae Infecting Eastern Honey Bee Workers

Simple SummaryAt present, interaction between Nosema ceranae and Apis cerana is poorly understood, though A. cerana is the original host for N. ceranae. Here, comparative investigation was conducted using transcriptome data from N. ceranae infecting Apis cerana cerana workers at seven days post inoculation (dpi) and 10 dpi (NcT1 and NcT2 groups) as well as N. ceranae spores (NcCK group). There were 1411, 604, and 38 DEGs identified in NcCK vs. NcT1, NcCK vs. NcT2, and NcT1 vs. NcT2 comparison groups. Additionally, 10 upregulated genes and nine downregulated ones were shared by above-mentioned comparison groups. GO classification and KEGG pathway analysis suggested that these DEGs were engaged in a number of key functional terms and pathways such as cell part and glycolysis. Further analysis indicated that most of virulence factor-encoding genes were upregulated, while a few were downregulated during the fungal infection. Findings in this current work provide a basis for clarifying the molecular mechanism udnerlying N. ceranae infection and host-microsporidian interaction during bee nosemosis.Apis cerana is the original host for Nosema ceranae, a widespread fungal parasite resulting in honey bee nosemosis, which leads to severe losses to the apiculture industry throughout the world. However, knowledge of N. ceranae infecting eastern honey bees is extremely limited. Currently, the mechanism underlying N. ceranae infection is still largely unknown. Based on our previously gained high-quality transcriptome datasets derived from N. ceranae spores (NcCK group), N. ceranae infecting Apis cerana cerana workers at seven days post inoculation (dpi) and 10 dpi (NcT1 and NcT2 groups), comparative transcriptomic investigation was conducted in this work, with a focus on virulence factor-associated differentially expressed genes (DEGs). Microscopic observation showed that the midguts of A. c. cerana workers were effectively infected after inoculation with clean spores of N. ceranae. In total, 1411, 604, and 38 DEGs were identified from NcCK vs. NcT1, NcCK vs. NcT2, and NcT1 vs. NcT2 comparison groups. Venn analysis showed that 10 upregulated genes and nine downregulated ones were shared by the aforementioned comparison groups. The GO category indicated that these DEGs were involved in a series of functional terms relevant to biological process, cellular component, and molecular function such as metabolic process, cell part, and catalytic activity. Additionally, KEGG pathway analysis suggested that the DEGs were engaged in an array of pathways of great importance such as metabolic pathway, glycolysis, and the biosynthesis of secondary metabolites. Furthermore, expression clustering analysis demonstrated that the majority of genes encoding virulence factors such as ricin B lectins and polar tube proteins displayed apparent upregulation, whereas a few virulence factor-associated genes such as hexokinase gene and 6-phosphofructokinase gene presented downregulation during the fungal infection. Finally, the expression trend of 14 DEGs was confirmed by RT-qPCR, validating the reliability of our transcriptome datasets. These results together demonstrated that an overall alteration of the transcriptome of N. ceranae occurred during the infection of A. c. cerana workers, and most of the virulence factor-related genes were induced to activation to promote the fungal invasion. Our findings not only lay a foundation for clarifying the molecular mechanism underlying N. ceranae infection of eastern honey bee workers and microsporidian–host interaction.

Sphaeromyxa azevedoi n. sp. (Myxozoa: Sphaeromyxidae) infecting the gall bladder of Gobioides grahamae (Perciformes: Gobiidae) in the Amazon region.

Sphaeromyxa azevedoi n. sp. is described from the gall bladder of the goby Gobioides grahamae (Gobiidae) captured on the Paracauari River in Salvaterra, on Marajó Island, northern Brazil. A total of 50 G. grahamae specimens were analysed, and 15 (30%) were parasitised by the plasmodia and myxospore of Sphaeromyxa azevedoi n. sp. Large plasmodia were observed floating in the bile. These plasmodia were flat, rounded, oval or elongated, and of varying sizes. The mature myxospores, found singly or in pairs, were 27.1 ± 2.7 (20.5-30.1) μmLength and 3.8 ± 0.2 (3.5-4.4) μmWidth in the valvular view. The myxospore has two polar capsules of equal size, 8.1 ± 0.6 (7.4-9.4) μm in length and 2.9 ± 0.2 (2.3-3.3) μm in width. A polar tubule was observed in each capsule, arranged perpendicularly to the principal axis, with three or four coils. The histological analysis showed that the plasmodia and myxospore are located in the lumen of the gall bladder, arranged in pairs, and the epithelium of the gall bladder presented multifocal necrosis. The SSU rDNA of Sphaeromyxa azevedoi n. sp. clusters in the 'balbianii' group of the Sphaeromyxa clade. The morphological characteristics and molecular phylogeny of Sphaeromyxa azevedoi n. sp. support its classification as a new species of the genus Sphaeromyxa, which represents an important advancement in the understanding of the diversity of the myxozoan parasite fauna of Brazilian fishes, especially considering that the new species may be detrimental to the host, a commercially important Brazilian fish species.

New data on Henneguya postexilis Minchew, 1977, a parasite of channel catfish Ictalurus punctatus, with notes on resolution of molecular markers for myxozoan phylogeny.

Previous morphological and histological data are supplemented with molecular and ultrastructural data for a Henneguya sp. isolated from farm-raised channel catfish Ictalurus punctatus in Mississippi, USA. Myxospores were cryptic, encapsulated within a thin layer of epithelium in the gill lamellae with spore measurements consistent with the original description of Henneguya postexilis Minchew, 1977. Myxospores were 42.7-49.1 µm in total length with spore bodies 12.1-17.2 × 3.6-4.8 × 2.9-3 µm. Polar capsules were of unequal length, with the longer capsule being 4.4-6.7 × 1.1-1.6 µm and the shorter capsule being 4.4-6.4 × 1.1-1.6 µm. Polar tubules had 6-8 turns. Caudal processes were 25.7-38.1 µm in length. Spores were encapsulated in a thin layer of epithelium in the gill lamellae. Molecular data from the most commonly used markers for myxozoan identification and phylogeny, partial 18S small subunit ribosomal gene (SSU), partial 28S large subunit ribosomal gene (LSU), and elongation factor 2 (EF2) were generated for H. postexilis. Additionally, novel data for LSU and EF2 were generated for archived myxozoan specimens from farm-raised catfish (H. mississippiensis, H. ictaluri, H. exilis, H. adiposa, H. sutherlandi, H. bulbosus, Unicauda fimbrethilae), as well as archived specimens from wild fish (H. laseeae [from Pylodictis olivaris], Hennegoides flockae [from Aphredoderus sayanus], Myxobolus cloutmani [from Cycleptus elongatus]. These include the first EF2 sequence data for the genera Hennegoides and Unicauda. Phylogenetic analyses using these data placed H. postexilis in well supported clades with other ictalurid-infecting Henneguya species. Phylogenetic signal assessments on these analyses suggest that while SSU provided the greatest phylogenetic signal, LSU yielded comparable signal, supporting previous work implying this region may be underutilised in reconstructing myxobolid phylogenies.

Mechanics of Microsporidian Polar Tube Firing.

As obligate intracellular parasites with reduced genomes, microsporidia must infect host cells in order to replicate and cause disease. They can initiate infection by utilizing a harpoon-like invasion organelle called the polar tube (PT). The PT is both visually and functionally a striking organelle and is a characteristic feature of the microsporidian phylum. Outside the host, microsporidia exist as transmissible, single-celled spores. Inside each spore, the PT is arranged as a tight coil. Upon germination, the PT undergoes a large conformational change into a long, linear tube and acts as a tunnel for the delivery of infectious cargo from the spore to a host cell. The firing process is extremely rapid, occurring on a millisecond timescale, and the emergent tube may be as long as 20 times the size of the spore body. In this chapter, we discuss what is known about the structure of the PT, the mechanics of the PT firing process, and how it enables movement of material from the spore body.

The Function and Structure of the Microsporidia Polar Tube.

Microsporidia are obligate intracellular pathogens that were initially identified about 160years ago. Current phylogenetic analysis suggests that they are grouped with Cryptomycota as a basal branch or sister group to the fungi. Microsporidia are found worldwide and can infect a wide range of animals from invertebrates to vertebrates, including humans. They are responsible for a variety of diseases once thought to be restricted to immunocompromised patients but also occur in immunocompetent individuals. The small oval spore containing a coiled polar filament, which is part of the extrusion and invasion apparatus that transfers the infective sporoplasm to a new host, is a defining characteristic of all microsporidia. When the spore becomes activated, the polar filament uncoils and undergoes a rapid transition into a hollow tube that will transport the sporoplasm into a new cell. The polar tube has the ability to increase its diameter from approximately 100nm to over 600nm to accommodate the passage of an intact sporoplasm and penetrate the plasmalemma of the new host cell. During this process, various polar tube proteins appear to be involved in polar tube attachment to host cell and can interact with host proteins. These various interactions act to promote host cell infection.