Empirical evidence that metabolic theory describes the temperature dependency of within-host parasite dynamics.

The complexity of host-parasite interactions makes it difficult to predict how host-parasite systems will respond to climate change. In particular, host and parasite traits such as survival and virulence may have distinct temperature dependencies that must be integrated into models of disease dynamics. Using experimental data from Daphnia magna and a microsporidian parasite, we fitted a mechanistic model of the within-host parasite population dynamics. Model parameters comprising host aging and mortality, as well as parasite growth, virulence, and equilibrium abundance, were specified by relationships arising from the metabolic theory of ecology. The model effectively predicts host survival, parasite growth, and the cost of infection across temperature while using less than half the parameters compared to modeling temperatures discretely. Our results serve as a proof of concept that linking simple metabolic models with a mechanistic host-parasite framework can be used to predict temperature responses of parasite population dynamics at the within-host level.

Development of Anncaliia algerae (Microsporidia) in Drosophila melanogaster.

Representatives of the genus Anncaliia are known as natural parasites of dipteran and coleopteran insects, amphipod crustaceans, but also humans, primarily with immunodeficiency. Anncaliia algerae-caused fatal myositis is considered as an emergent infectious disease in humans. A. (=Nosema, Brachiola) algerae, the best studied species of the genus, demonstrates the broadest among microsporidia range of natural and experimental hosts, but it has never been propagated in Drosophila. We present ultrastructural analysis of development of A. algerae in visceral muscles and adipocytes of Drosophila melanogaster 2 wk after per oral experimental infection. We observed typical to Anncaliia spp. features of ultrastructure and cell pathology including spore morphology, characteristic extensions of the plasma membrane, and presence of "ridges" and appendages of tubular material at proliferative stages. Anncaliia algerae development in D. melanogaster was particularly similar to one of A. algerae and A.(Brachiola) vesicularum in humans with acute myositis. Given D. melanogaster is currently the most established genetic model, with a fully sequenced genome and easily available transgenic forms and genomic markers, a novel host-parasite system might provide new genetic tools to investigate host-pathogen interactions of A. algerae, as well to test antimicrosporidia drugs.

Host genotype and environment affect the trade-off between horizontal and vertical transmission of the parasite Edhazardia aedis.

BACKGROUND: If a parasite is able to transmit horizontally or vertically, which transmission mode will it choose? We investigated how the growth conditions and the genotype of the mosquito Aedes aegypti affect the transmission mode of the parasite Edhazardia aedis. RESULTS: In poor conditions the parasites were more likely to be transmitted horizontally, whereas in favourable conditions they were more likely to be transmitted vertically. Unfavourable conditions delayed emergence, giving the parasite more time to produce its horizontally transmitted stage; in more favourable conditions mosquitoes have greater reproductive success, increasing the effectiveness of vertical transmission. In addition, the parasite's ability to transmit vertically was influenced by the genetic background of the host (i.e., its full-sib family), giving a genetic correlation between the host's life-history and which of the parasite's transmission mode it enables. In particular, genotypes with large bodies (and therefore high fecundity) were more likely to enable vertical transmission than genotypes with small bodies. This led to a trade-off among the host's families (which can be interpreted as a genetic correlation) for the parasite's transmission mode. CONCLUSIONS: Since horizontal transmission is linked to higher virulence than vertical transmission, the host's contribution to transmission mode has important consequences for the evolution of parasites with mixed-mode transmission.

The roles of microsporidia spore wall proteins in the spore wall formation and polar tube anchorage to spore wall during development and infection processes.

Microsporidia are highly specialized obligate intracellular, spore forming divergent fungi with a wide variety host range that includes most vertebrates and invertebrates. The resistant spores are surrounded by a rigid cell wall which consists of three layers: the electron-lucent chitin and protein inner endospore, the outer-electron-dense and mainly proteinaceous exospore and plasma membrane. Interestingly, microsporidia owns a special invasion organelle, called polar tube, coiled within the interior of the spore wall and attached to anchoring disk at the anterior end of spore. Spore wall and polar tube are the major apparatuses for mature spores adhering and infecting to the host cells. In this review, we summarize the research advances in spore wall proteins (SWPs) related to spore adherence and infection, and SWPs and deproteinated chitin spore coats (DCSCs) interaction associated with SWPs deposit processes and spore wall assembly. Furthermore, we highlight the SWPs-polar tube proteins (PTPs) interaction correlated to polar tube orderly orientation, arrangement and anchorage to anchoring disk. Based on results obtained, it is helpful to improve understanding of the spore wall assembly and polar tube orderly arrangement mechanisms and molecular pathogenesis of microsporidia infection. Also, such information will provide a basis for developing effective control strategies against microporidia.

A Novel Spore Wall Protein from Antonospora locustae (Microsporidia: Nosematidae) Contributes to Sporulation.

Microsporidia are obligate intracellular parasites, existing in a wide variety of animal hosts. Here, we reported AlocSWP2, a novel protein identified from the spore wall of Antonospora locustae (formerly, Nosema locustae, and synonym, Paranosema locustae), containing four cysteines that are conserved among the homologues of several Microspodian pathogens in insects and mammals. AlocSWP2 was detected in the wall of mature spores via indirect immunofluorescence assay. In addition, immunocytochemistry localization experiments showed that the protein was observed in the wall of sporoblasts, sporonts, and meronts during sporulation within the host body, also in the wall of mature spores. AlocSWP2 was not detected in the fat body of infected locust until the 9th day after inoculating spores via RT-PCR experiments. Furthermore, the survival percentage of infected locusts injected with dsRNA of AlocSWP2 on the 15th, 16th, and 17th days after inoculation with microsporidian were significantly higher than those of infected locusts without dsRNA treatment. Conversely, the amount of spores in locusts infected with A. locustae after treated with RNAi AlocSWP2 was significantly lower than those of infected locusts without RNAi of this gene. This novel spore wall protein from A. locustae may be involved in sporulation, thus contributing to host mortality.

The small GTPase RAB-11 directs polarized exocytosis of the intracellular pathogen N. parisii for fecal-oral transmission from C. elegans.

Pathogen exit is a key stage in the spread and propagation of infectious disease, with the fecal-oral route being a common mode of disease transmission. However, it is poorly understood which molecular pathways provide the major modes for intracellular pathogen exit and fecal-oral transmission in vivo. Here, we use the transparent nematode Caenorhabditis elegans to investigate intestinal cell exit and fecal-oral transmission by the natural intracellular pathogen Nematocida parisii, which is a recently identified species of microsporidia. We show that N. parisii exits from polarized host intestinal cells by co-opting the host vesicle trafficking system and escaping into the lumen. Using a genetic screen, we identified components of the host endocytic recycling pathway that are required for N. parisii spore exit via exocytosis. In particular, we show that the small GTPase RAB-11 localizes to apical spores, is required for spore-containing compartments to fuse with the apical plasma membrane, and is required for spore exit. In addition, we find that RAB-11-deficient animals exhibit impaired contagiousness, supporting an in vivo role for this host trafficking factor in microsporidia disease transmission. Altogether, these findings provide an in vivo example of the major mode of exit used by a natural pathogen for disease spread via fecal-oral transmission.

Morphological characterization and HSP70-, IGS-based phylogenetic analysis of two microsporidian parasites isolated from Antheraea pernyi.

Two microsporidian isolates were extracted from single infected egg-laying tussah silk moth (Antheraea pernyi) in Liaoning Province, China. The microsporidia were subsequently grown in silk moth larvae, isolated, and subjected to morphological characterization (by light and transmission electron microscopy) and phylogenetic analysis (based on conserved genes). One type of spore was long-axis-oval in shape, measuring 4.71 × 1.95 µm, and the other type was short-axis-oval, measuring 3.64 × 2.17 µm. These dimensions were markedly different from those reported in the spores of the common microsporidia infecting A. pernyi, namely, Nosema pernyi (4.36 × 1.49 µm). A neighbor-joining phylogenetic tree based on HSP70 indicated that these microsporidia belonged to Nosema species and were closely related with Nosema bombycis and Nosema ceranae. Furthermore, in the phylogenetic tree based on the intergenic spacer (IGS) region, the long-axis-oval isolates were closely related and tended to form a clade away from the short-axis-oval isolates and N. pernyi isolates. The microsporidia isolated from A. pernyi clustered in one group. Nosema bombycis, Nosema spodopterae, and Endoreticulatus spp. appeared to be genetically distant from N. pernyi. The two isolates from A. pernyi fell in the Nosema group, but their spores differed from those of the spores of the common A. pernyi parasite N. pernyi, both in morphological and genetic aspects. The two isolates were designated Nosema sp. Ap (L) and Nosema sp. Ap (S). IGS was found to be informative in ascertaining phylogenetic relationships among species, and even closely related strains, of microsporidia.

Transcriptomic profiling of host-parasite interactions in the microsporidian Trachipleistophora hominis.

BACKGROUND: Trachipleistophora hominis was isolated from an HIV/AIDS patient and is a member of a highly successful group of obligate intracellular parasites. METHODS: Here we have investigated the evolution of the parasite and the interplay between host and parasite gene expression using transcriptomics of T. hominis-infected rabbit kidney cells. RESULTS: T. hominis has about 30% more genes than small-genome microsporidians. Highly expressed genes include those involved in growth, replication, defence against oxidative stress, and a large fraction of uncharacterised genes. Chaperones are also highly expressed and may buffer the deleterious effects of the large number of non-synonymous mutations observed in essential T. hominis genes. Host expression suggests a general cellular shutdown upon infection, but ATP, amino sugar and nucleotide sugar production appear enhanced, potentially providing the parasite with substrates it cannot make itself. Expression divergence of duplicated genes, including transporters used to acquire host metabolites, demonstrates ongoing functional diversification during microsporidian evolution. We identified overlapping transcription at more than 100 loci in the sparse T. hominis genome, demonstrating that this feature is not caused by genome compaction. The detection of additional transposons of insect origin strongly suggests that the natural host for T. hominis is an insect. CONCLUSIONS: Our results reveal that the evolution of contemporary microsporidian genomes is highly dynamic and innovative. Moreover, highly expressed T. hominis genes of unknown function include a cohort that are shared among all microsporidians, indicating that some strongly conserved features of the biology of these enormously successful parasites remain uncharacterised.

Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth.

Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungal-related parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and Nematocida sp1, which are natural pathogens of Caenorhabditis nematodes and provide model systems for studying microsporidian pathogenesis. We performed deep sequencing of transcripts from a time course of N. parisii infection. Examination of pathogen gene expression revealed compact transcripts and a dramatic takeover of host cells by Nematocida. We also performed phylogenomic analyses of Nematocida and other microsporidian genomes to refine microsporidian phylogeny and identify evolutionary events of gene loss, acquisition, and modification. In particular, we found that all microsporidia lost the tumor-suppressor gene retinoblastoma, which we speculate could accelerate the parasite cell cycle and increase the mutation rate. We also found that microsporidia acquired transporters that could import nucleosides to fuel rapid growth. In addition, microsporidian hexokinases gained secretion signal sequences, and in a functional assay these were sufficient to export proteins out of the cell; thus hexokinase may be targeted into the host cell to reprogram it toward biosynthesis. Similar molecular changes appear during formation of cancer cells and may be evolutionary strategies adopted independently by microsporidia to proliferate rapidly within host cells. Finally, analysis of genome polymorphisms revealed evidence for a sexual cycle that may provide genetic diversity to alleviate problems caused by clonal growth. Together these events may explain the emergence and success of these diverse intracellular parasites.

Early development and tissue distribution of Pseudoloma neurophilia in the zebrafish, Danio rerio.

The early proliferative stages of the microsporidian parasite, Pseudoloma neurophilia were visualized in larval zebrafish, Danio rerio, using histological sections with a combination of an in situ hybridization probe specific to the P. neurophilia small-subunit ribosomal RNA gene, standard hematoxylin-eosin stain, and the Luna stain to visualize spores. Beginning at 5 d post fertilization, fish were exposed to P. neurophilia and examined at 12, 24, 36, 48, 72, 96, and 120 h post exposure (hpe). At 12 hpe, intact spores in the intestinal lumen and proliferative stages developing in the epithelial cells of the anterior intestine and the pharynx and within hepatocytes were observed. Proliferative stages were visualized in the pancreas and kidney at 36-48 hpe and in the spinal cord, eye, and skeletal muscle beginning at 72 hpe. The first spore stages of P. neurophilia were observed at 96 hpe in the pharyngeal epithelium, liver, spinal cord, and skeletal muscle. The parasite was only observed in the brain of larval fish at 120 hpe. The distribution of the early stages of P. neurophilia and the lack of mature spores until 96 hpe indicates that the parasite gains access to organs distant from the initial site of entry, likely by penetrating the intestinal wall with the polar tube.

Description of Metchnikovella spiralis sp. n. (Microsporidia: Metchnikovellidae), with notes on the ultrastructure of metchnikovellids.

The present paper reports results of a transmission electron microscopy study of a new metchikovellid microsporidium. It was isolated from gregarines Polyrhabdina sp. inhabiting guts of polychaetes Pygospio elegans sampled at the White Sea silt littoral zone. Free sporogony (FS) occurred in the life cycle of the microsporidium alongside sac-bound sporogony (BS). Free spores resided in a parasitophorous vacuole and were of typical metchnikovellidean structure, uninucleate and oblong. They measured on sections 2·0-3·2×1·3-1·9 µm. The life cycle included pre-sporogonial stages represented by dikaryotic cells and 4-nucleate cells with coupled nuclei. A multinucleate sporogonial plasmodium of FS split in numerous (>10) sporoblasts. In BS segregation of sporoblasts occurred within thick-walled cysts by internal budding. Spore sacs of this microsporidium, measuring on average 11·6×4·7 µm, were limited by a thick electron-dense wall, externally ornamented with spirally wound cords of dense material. These oval spore sacs contained eight barrel-shaped spores, comparable in size and ultrastructure to FS spores. Ultrastructure of both types of spores and intracellular development of the new microsporidium and Metchnikovella spp. were similar, suggesting they belong to the same genus. In this paper we describe a new species Metchnikovella spiralis and discuss morphology of metchnikovellids in the context of putative evolutionary history of Microsporidia.

In vitro growth of the microsporidian Heterosporis saurida in the eel kidney EK-1 cell line.

Heterosporis saurida is an intracellular microsporidian that infects lizardfish Saurida undosquamis. Although some attempts have been introduced to clarify microsporidian host-pathogen interactions, development of novel strategies to combat fish diseases is still needed. Here we present an in vitro cultivation model for fish microsporidia based on an eel kidney cell line (EK-1), which is susceptible to infection by H. saurida. Spores were isolated from infected lizardfish and used to inoculate EK-1 cells. H. saurida were propagated in the eel kidney EK-1 cell line and detected by immunofluorescence. Developmental stages of H. saurida were seen in EK-1 cells by transmission electron microscopy. Identity of the parasite was confirmed by partial sequencing of the 16S rDNA gene. Our cell culture model provides a valuable means to explore molecular and immunological events and will facilitate development of effective treatment strategies.

Cytological, molecular and life cycle characterization of Anostracospora rigaudi n. g., n. sp. and Enterocytospora artemiae n. g., n. sp., two new microsporidian parasites infecting gut tissues of the brine shrimp Artemia.

Two new microsporidia, Anostracospora rigaudi n. g., n. sp., and Enterocytospora artemiae n. g., n. sp. infecting the intestinal epithelium of Artemia parthenogenetica Bowen and Sterling, 1978 and Artemia franciscana Kellogg, 1906 in southern France are described. Molecular analyses revealed the two species belong to a clade of microsporidian parasites that preferentially infect the intestinal epithelium of insect and crustacean hosts. These parasites are morphologically distinguishable from other gut microsporidia infecting Artemia. All life cycle stages have isolated nuclei. Fixed spores measure 1·3×0·7 µm with 5-6 polar tube coils for A. rigaudi and 1·2×0·9 µm with 4 polar tube coils for E. artemiae. Transmission of both species is horizontal, most likely through the ingestion of spores released with the faeces of infected hosts. The minute size of these species, together with their intestinal localization, makes their detection and identification difficult. We developed two species-specific molecular markers allowing each type of infection to be detected within 3-6 days post-inoculation. Using these markers, we show that the prevalence of these microsporidia ranges from 20% to 75% in natural populations. Hence, this study illustrates the usefulness of molecular approaches to study prevalent, but cryptic, infections involving microsporidian parasites of gut tissues.

Ovavesicula popilliae (Microsporidia: Ovavesiculidae) spore production in naturally infected adult Japanese beetles (Coleoptera: Scarabaeidae).

Ovavesicula popilliae is a microsporidian that infects both Japanese beetle larvae and adults. This is the first study quantifying the number of O. popilliae spores produced by Japanese beetle adults. Mean spore production per adult Japanese beetle was 2.67 × 10(7) (SE ± 4.65 × 10(6)) spores with a range of 1.46 × 10(6)-1.02 × 10(8). The number of spores produced per host is similar to other microsporidian species and may help explain the speed with which this pathogen has spread from introduction sites to surrounding areas.

Splicing and transcription differ between spore and intracellular life stages in the parasitic microsporidia.

Microsporidia are a diverse group of highly derived fungal relatives that are intracellular parasites of many animals. Both transcription and introns have been shown to be unusual in microsporidia: The complete genome of the human parasite Encephalitozoon cuniculi has only a few very short introns, and two distantly related microsporidian spores have been shown to harbor transcripts encoding several genes that overlap on different strands. However, microsporidia alternate between two life stages: the intracellular proliferative stage and the extracellular and largely metabolically dormant infectious spore. To date, most studies have focused on the spore. Here, we have compared transcription profiles for a number of genes from both life stages of microsporidia and found major differences in both the prevalence of overlapping transcription and splicing. Specifically, spore transcripts in E. cuniculi have longer 5' untranslated regions, overlap more frequently with upstream genes, and have a significantly higher number of transcription initiation sites compared with intracellular transcripts from the same species. In addition, we demonstrate that splicing occurs exclusively in the intracellular stage and not in spore messenger RNAs (mRNAs) in both E. cuniculi and the distantly related Antonospora locustae. These differences between the microsporidian life stages raise questions about the functional importance of transcripts in the spore. We hypothesize that at least some transcripts in spores are a product of the cell's transition into a dormant state and that these unusual mRNAs could play a structural role rather than an informational one.

A cell culture model for Nosema ceranae and Nosema apis allows new insights into the life cycle of these important honey bee-pathogenic microsporidia.

The population of managed honey bees has been dramatically declining in the recent past in many regions of the world. Consensus now seems to be that pathogens and parasites (e.g. the ectoparasitic mite Varroa destructor, the microsporidium Nosema ceranae and viruses) play a major role in this demise. However, little is known about host-pathogen interactions for bee pathogens and attempts to develop novel strategies to combat bee diseases have been hampered by this gap in our knowledge. One reason for this dire situation is the complete lack of cell cultures for the propagation and study of bee pathogens. Here we present a cell culture model for two honey bee-pathogenic microsporidian species, Nosema apis and N. ceranae. Our cell culture system is based on a lepidopteran cell line, which proved to be susceptible to infection by both N. ceranae and N. apis and enabled us to illustrate the entire life cycle of these microsporidia. We observed hitherto undescribed spindle-shaped meronts and confirmed our findings in infected bees. Our cell culture model provides a previously unavailable means to explore the nature of interactions between the honey bee and its pathogen complex at a mechanistic level and will allow the development of novel treatment strategies.

Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. infecting invasive Chinese mitten crabs (Eriocheir sinensis) in Europe.

We describe a microsporidian parasite infecting non-native Chinese mitten crabs (Eriochier sinensis) from Europe. Electron microscopy revealed merogonic and sporogonic life stages bound within a plasmalemma. The crab parasite develops polar tube precursors at the sporont stage but does not complete formation of the intact spore extrusion apparatus at the stage of the sporogonial plasmodium like Enterocytozoon bienuesi and other representatives of the Enterocytozoonidae. Its presence within an aquatic crustacean host, and a distinct molecular phylogeny based on partial small subunit ribosomal RNA (SSU rRNA) gene sequences also place it relatively close, though distinct to, existing genera within the Enterocytozoonidae. Consideration of morphological and phylogenetic characteristics of other hepatopancreas-infecting microsporidia from crustaceans suggests that certain ones (e.g. Enterospora canceri) are retained within the clade corresponding to the existing family Enterocytozoonidae, while others, including the parasite described here, may eventually be grouped in a sister taxon potentially of family rank. Based upon morphological and host similarity, it is likely that the parasite described here is the same as Endoreticulatus eriocheir (Wang and Chen, 2007), previously described from Chinese mitten crabs in Asia. However, using a combined taxonomic approach based upon morphological and phylogenetic data, we propose the formation of a new genus (Hepatospora) to replace the previous generic classification of the Asian parasite as Endoreticulatus. The microsporidian from the hepatopancreas of E. sinensis is named Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. It is assumed that the parasite was introduced during initial invasions of this crab to Europe during the early 20th Century.

Cost of co-infection controlled by infectious dose combinations and food availability.

To what extent the combined effect of several parasite species co-infecting the same host (i.e. polyparasitism) affects the host's fitness is a crucial question of ecological parasitology. We investigated whether the ecological setting can influence the co-infection's outcome with the mosquito Aedes aegypti and two parasites: the microsporidium Vavraia culicis and the gregarine Ascogregarina culicis. The cost of being infected by the two parasites depended on the interaction between the two infectious doses and host food availability. The age at pupation of the mosquito was delayed most when the doses of the two parasites were highest and little food was available. As infectious dose increases with the parasites' prevalence and intensity of transmission, the cost of being co-infected depends on the epidemiological status of the two parasite species.

Budding: a new stage in the development of Chytridiopsis typographi (Zygomycetes: Microsporidia).

Chytridiopsis typographi Weiser, 1954, the microsporidian pathogen of the spruce bark beetle, Ips typographus L. (Coleoptera: Scolytidae), has an early developmental period with plurinucleate mother cells, each of which produces a single bud. The globular bud is connected with the mother cell by a collar and the cellular constituents are pushed to the distant end of the bud. Both the mother cell and the bud continue to develop; the bud then separates from the mother cell and grows to produce a cell of the same type. Both cells then continue sporogonial development and produce sporophorous vesicles with 16-32 spores. The process of a single mother cell producing a single bud that grows to an identical stage is new in the development of C. typographi and has no analogy in other Microsporidia.

Propagation of human enteropathogens in constructed horizontal wetlands used for tertiary wastewater treatment.

Constructed subsurface flow (SSF) and free-surface flow (FSF) wetlands are being increasingly implemented worldwide into wastewater treatments in response to the growing need for microbiologically safe reclaimed waters, which is driven by an exponential increase in the human population and limited water resources. Wastewater samples from four SSF and FSF wetlands in northwestern Ireland were tested qualitatively and quantitatively for Cryptosporidium spp., Giardia duodenalis, and human-pathogenic microsporidia, with assessment of their viability. Overall, seven species of human enteropathogens were detected in wetland influents, vegetated areas, and effluents: Cryptosporidium parvum, C. hominis, C. meleagridis, C. muris, G. duodenalis, Encephalitozoon hellem, and Enterocytozoon bieneusi. SSF wetland had the highest pathogen removal rate (i.e., Cryptosporidium, 97.4%; G. duodenalis, 95.4%); however, most of these values for FSF were in the negative area (mean, -84.0%), meaning that more pathogens were discharged by FSF wetlands than were delivered to wetlands with incoming wastewater. We demonstrate here that (i) the composition of human enteropathogens in wastewater entering and leaving SSF and FSF wetlands is highly complex and dynamic, (ii) the removal and inactivation of human-pathogenic microorganisms were significantly higher at the SSF wetland, (iii) FSF wetlands may not always provide sufficient remediation for human enteropathogens, (iv) wildlife can contribute a substantial load of human zoonotic pathogens to wetlands, (v) most of the pathogens discharged by wetlands were viable, (vi) large volumes of wetland effluents can contribute to contamination of surface waters used for recreation and drinking water abstraction and therefore represent a serious public health threat, and (vii) even with the best pathogen removal rates achieved by SSF wetland, the reduction of pathogens was not enough for a safety reuse of the reclaimed water. To our knowledge, this is the first report of C. meleagridis from Ireland.