A Novel Fluorescent Labeling Method Enables Monitoring of Spatio-Temporal Dynamics of Developing Microsporidia.

The microsporidium, Anncaliia algerae (Brachiola algerae), is a eukaryotic obligate intracellular parasite first isolated from mosquitoes and is an important opportunistic human pathogen that can cause morbidity and mortality among immune-compromised individuals including patients with AIDS and those undergoing chemotherapy. There is little known about the Microsporidia-host cell interface in living host cells, due to current approaches being limited by the lack of fluorescent reporters for detecting the parasite lifecycle. Here, we have developed and applied novel vital fluorescent parasite labeling methodologies in conjunction with fluorescent protein-tagged reporters to track simultaneously the dynamics of both parasite and host cell specific components, including the secretory and endocytic trafficking pathways, during the entire infection time period. We have found dramatic changes in the dynamics of host secretory trafficking organelles during the course of infection. The Golgi compartment is gradually disassembled and regenerated into mini-Golgi structures in parallel with cellular microtubule depolymerization. Importantly, we find that Microsporidia progeny are associated with these de novo formed mini-Golgi structures. These host structures appear to create a membrane bound niche environment for parasite development. Our studies presented here provide novel imaging tools and methodologies that will facilitate in understanding the biology of microsporidial parasites in the living host.

Development, ultrastructural pathology, and taxonomic revision of the Microsporidial genus, Pseudoloma and its type species Pseudoloma neurophilia, in skeletal muscle and nervous tissue of experimentally infected zebrafish Danio rerio.

The microsporidium Pseudoloma neurophilia was initially reported to infect the central nervous system of zebrafish causing lordosis and eventually death. Subsequently, muscle tissue infections were also identified. To understand the infection process, development, and ultrastructural pathology of this microsporidium, larval and adult zebrafish were fed P. neurophilia spores. Spores were detected in the larval fish digestive tract 3-h postexposure (PE). By 4.5-d PE, developing parasite stages were identified in muscle tissue. Wet preparations of larvae collected at 8-d PE showed aggregates of spores in the spinal cord adjacent to the notochord. All parasite stages, including spores, were present in the musculature of larval fish 8-d PE. Adult zebrafish sacrificed 45-d PE had fully developed infections in nerves. Ultrastructural study of the developmental cycle of P. neurophilia revealed that proliferative stages undergo karyokinesis, producing tetranucleate stages that then divide into uninucleate cells. The plasmalemma of proliferative cells has a previously unreported glycocalyx-like coat that interfaces with the host cell cytoplasm. Sporogonic stages form sporophorous vacuoles (SPOV) derived from the plasmalemmal dense surface coat, which "blisters" off sporonts. Uninucleate sporoblasts and spores develop in the SPOV. The developmental cycle is identical in both nerve and muscle. The SPOV surface is relatively thick and is the outermost parasite surface entity; thus, xenomas are not formed. Based on the new information provided by this study, the taxonomic description of the genus Pseudoloma and its type species, P. neurophilia, is modified and its life cycle described.

Life cycle, ultrastructure, and molecular phylogeny of Crispospora chironomi g.n. sp.n. (Microsporidia: Terresporidia), a parasite of Chironomus plumosus L. (Diptera: Chironomidae).

The life cycle, ultrastructure, and molecular phylogeny of a new microsporidium Crispospora chironomi g.n. sp.n., a parasite of the midge Chironomus plumosus, are described. The parasite infects the gut epithelium of the host larvae and possesses sporogonies of two types, polysporoblastic and disporoblastic, respectively, proceeding within the same host cell. In the sporogonial sequence of the first type, dozens of spherical monokaryotic spores within a thick-walled capsule are formed. The spores are 1.5-2.0 µm in diameter; the exospore possesses two to three bundles of tubular protrusions. In the sporogonial sequence of the second type, diplokaryotic oval spores, 2.5 × 1.5 µm in size, are formed within a compartment, partially surrounded with multilayered membranes. Spores of both types are similar in respect to inner structure, possessing a well-developed extrusion apparatus with (a) the anterior vesicular part of the polaroplast covering the lamellar posterior one and (b) isofilar polar filament with several coils in one row. Small subunit ribosomal DNA phylogeny showed position of the new microsporidium in a cluster uniting microsporidia of terrestrial origin infecting diverse hosts, nested within Clade IV, corresponding to Class Terresporidia sensu Vossbrinck and Debrunner-Vossbrinck (Folia Parasitol 52:131-142, 2005).

Biology and life-cycle of the microsporidium Kneallhazia solenopsae Knell Allan Hazard 1977 gen. n., comb. n., from the fire ant Solenopsis invicta.

Thelohania solenopsae is a unique microsporidium with a life-cycle finely tuned to parasitizing fire ant colonies. Unlike other microsporidia of social hymenopterans, T. solenopsae infects all castes and stages of the host. Four distinctive spore types are produced: diplokaryotic spores, which develop only in brood (Type 1 DK spores); octets of octospores within sporophorous vesicles, the most prominent spore type in adults but never occurring in brood; Nosema-like diplokaryotic spores (Type 2 DK spores) developing in adults; and megaspores, which occur occasionally in larvae 4, pupae, and adults of all castes but predominantly infect gonads of alates and germinate in inseminated ovaries of queens. Type 2 DK spores function in autoinfection of adipocytes. Proliferation of diplokaryotic meronts in some cells is followed by karyogamy of diplokarya counterparts and meiosis, thereby switching the diplokaryotic sequence to octospore or megaspore development. Megaspores transmit the pathogen transovarially. From the egg to larvae 4, infection is inapparent and can be detected only by PCR. Type 1 DK spore and megaspore sequences are abruptly triggered in larvae 4, the key stage in intra-colony food distribution via trophallaxis, and presumably the central player in horizontal transmission of spores. Molecular, morphological, ultrastructural and life-cycle data indicate that T. solenopsae must be assigned to a new genus. We propose a new combination, Kneallhazia solenopsae.

A new microsporidian infecting the musculature of the endangered tidewater goby (Gobiidae).

A previously unrecognized microsporidian (Kabatana newberryi n. sp.) is described from the musculature of Eucyclogobius newberryi (Gobiidae) in Big Lagoon, Humboldt County, California. Spores are ovoid, ranging in size from 2.8 +/- 0.3 microm in total length and 1.9 +/- 0.4 microm in width (measurements of 30 spores made by calculation from micrograph). The polar filament has 9-10 coils in 1-2 rows. Development occurs in direct contact with host muscle cell cytoplasm, without xenoma or sporophorous vesicle. Phylogenetic analysis of the new species and of 35 other microsporidians known to infect fish using 1115 base pairs of aligned 16S rRNA gene indicate the new species is most closely related to Kabatana takedai. However, the new species differs by 11% sequence divergence from K. takedai. Divergence in morphology and genetic data allow for diagnosis from all other fish-infecting microsporidia and supports recognition of a new species of microsporidian, Kabatana newberryi n. sp., presently known only from a suspected specific host, the endangered tidewater goby Eucyclogobius newberryi.

Development, ultrastructure, natural occurrence, and molecular characterization of Liebermania patagonica n. g., n. sp., a microsporidian parasite ot the grasshopper Tristira magellanica (Orthoptera: Tristiridae).

A new microsporidium, Liebermannia patagonica n. gen., n. sp., is described from midgut and gastric caecum epithelial cells of Tristira magellanica, an apterous grasshopper species of southern Patagonia, Argentina. L.patagonica is diplokaryotic, apansporoblastic, homosporous, and polysporoblastic. Transitional (from merogony to sporogony) stages and sporonts of L. patagonica were surrounded by host rough endoplasmic reticulum. The ovocylindrical spores measured 2.9 +/- 0.09 x 1.2 +/- 0.04 microm (fresh, n = 50), and they had an isofilar polar filament of only three coils and a cluster of tubules instead of a classical posterior vacuole. Prevalence was high (up to 80.6%) at the type locality for the four years sampled . Maximum likelihood , neighbor joining, maximum parismony analyses of the small submit rDNA all placed L.patagonica(Accession No. DQ 239917) in one with Orthosomella operophterae.

Electron microscopic study of a new microsporean Microsporidium epithelialis sp. n. infecting Tubifex sp. (Oligochaeta).

The cytology of a new microsporean parasite Microsporidium epithelialis sp. n. from the intestinal epithelial cells of the freshwater oligochaete Tubifex sp. (Tubificidae) is described. The microsporean occurred together with an actinosporean of the genus Triactinomyxon, which was found between the epithelial cells. The merogonic and sporogonic stages (mature spores included) of the microsporean parasite are monokaryotic. An individual sporophorous vesicle surrounds each spore. The fixed and stained spore has an average dimension of 1.9-2.5 x 0.9-1.2 microm. The spores are oval with a characteristic surface layer, showing ornamentation-like projections, which are in close contact to the exospore. A short polar filament forming three to four coils traverses the polaroplast with two lamellar layers. The ultrastructure and other characteristic features of this microsporean parasite are distinct from those of the microsporean species described so far from oligochaetes.