Fish microsporidia: fine structural diversity and phylogeny

Structural diversity of fish microsporidian life cycle stages and of the host-parasite interface is reviewed. In the infected cell of the fish host, microsporidia may either cause serious degradation of the cytoplasm and demise of the cell, or they may elicit host cell hypertrophy, producing a parasite-hypertrophic host cell complex, the xenoma. The structure of the xenoma and of its cell wall may differ according to the genus of the parasite, and seems to express properties of the parasite rather than those of the host. In merogony, the parasite cell surface interacts with the host cell in diverse ways, the most conspicuous being the production of thick envelopes of different types. Sporogony stages reveal different types of walls or membranes encasing the sporoblasts and later the spores and these envelopes may be of host or parasite origin. Nucleospora differs from all other fish microsporidia by its unique process of sporogony. Except for the formation of conspicuous xenomas, there are no essentially different structures in fish-infecting microsporidia compared with microsporidia from other hosts. Although the structures associated with the development of fish microsporidia cannot be attributed importance in tracing the phylogeny, they are relevant for practical determination and assessing the relation to the host. The possibility of the existence of an intermediate host is discussed. Higher-level classification of Microsporidia is briefly discussed and structure and evolutionary rates in microsporidian rDNA are reviewed. Discussion of rDNA molecular phylogeny of fish-infecting microsporidia is followed by classification of these parasites. Most form a rather cohesive clade. Outside this clade is the genus Nucleospora, separated at least at the level of Order. Within the main clade, however, there are six species infecting hosts other than fish. Based on data available for analysis, a tentative classification of fish-infecting microsporidia into five groups is proposed. Morphologically defined groups represent families, others are referred to as clades. Group 1, represented by family Pleistophoridae, includes Pleistophora, Ovipleistophora and Heterosporis; Vavraia and Trachipleistophora infect non-fish hosts. Group 2, represented by family Glugeidae, is restricted to genus Glugea and Tuzetia weidneri from crustaceans. Group 3 comprises three clades: Loma and a hyperparasitic microsporidian from a myxosporean; Ichthyosporidium and Pseudoloma clade and the Loma acerinae clade. For the latter species a new genus has to be established. Group 4 contains two families, Spragueidae with the genus Spraguea and Tetramicridae with genera Microgemma and Tetramicra, and the Kabatana and Microsporidium seriolae clade. Group 5 is represented by the family Enterocytozoonidae with the genus Nucleospora and mammal-infecting genus Enterocytozoon.     

Amazonspora hassar n. gen. and n. sp. (phylum Microsporidia,fam. Glugeidae), a parasite of the Amazonian teleost Hassar orestis (fam. Doradidae)

We describe the microsporidian Amazonspora hassar n. gen., n. sp. from the gill xenomas of the teleost Hassar orestis (Doradidae) collected in the estuarine region of the Amazon River. The parasite appeared as a small whitish xenoma located in the gill filaments near the blood vessels. Each xenoma consisted of a single hypertrophic host cell (HHC) in the cytoplasm of which the microsporidian developed and proliferated. The xenoma wall was composed of up to approximately 22 juxtaposed crossed layers of collagen fibers. The plasmalemma of the HHC presented numerous anastomosed, microvilli-like structures projecting outward through the 1-3 first internal layers of the collagen fibrils. The parasite was in direct contact with host cell cytoplasm in all stages of the cycle (merogony and sporogony). Sporogony appears to divide by plasmotomy, giving rise to 4 uninucleate sporoblasts, which develop into uninucleate spores. The ellipsoidal spores measured 2.69 +/- 0.45 x 1.78 +/- 0.18 microm, and the wall measured approximately 75 nm. The anchoring disk of the polar filament was subterminal, being shifted laterally from the anterior pole. The polar filament was arranged into 7-8 coils in a single layer in the posterior half of the spore, surrounding the posterior vacuole. The polaroplast surrounded the uncoiled portion of the polar filament, and it was exclusively lamellar. The spores and different life-cycle stages were intermingled within the cytoplasm of the HHC, surrounding the central hypertrophic deeply branched nucleus. The ultrastructural morphology of this microsporidian parasite suggests the erection of a new genus and species.     

Nosema helminthorum Moniez, 1887 (Microspora,Nosematidae):A Taxonomic Enigma1

ABSTRACT. The microsporidian parasite known as Nosema helminthorum Moniez, 1887, parasitic in the tapeworm Moniezia expansa (Rudolphi, 1810), has been shown by electron microscopy to have two cycles of development, one with isolated nuclei, the other with paired nuclei (diplokarya). Both merogony and sporogony of the two separate sequences take place in direct contact with the host cell cytoplasm and ultimately give rise to unikaryotic and diplokaryotic sporoblasts. Sporogony is disporoblastic. The nuclear condition of the spores was not seen. The sequences, corresponding to those of the genera Unikaryon and Nosema, may be part of a single dimorphic life cycle and, if so, the species will have to be transferred to a new genus.     

Ultrastructure of the Peripheral Zone of a Glugea-Induced Xenoma*

SYNOPSIS. The Glugea stephani-induced xenoma in the winter flounder, Pseudopleuronectes americanus, is a large spherical host-parasite complex, up to 4.0 mm in diameter, with the host and parasite components of the xenoma being most active in the peripheral zone. The xenoma has an extensive periodic acid-silver methenamine-positive surface coat covering the plasma membrane. The surface of this membrane is amplified by the presence of numerous folds and fine tubular extensions. The peripheral zone of the xenoma contains many host-cell mitochondria in addition to numerous microsporidan parasites. At the ultrastructural level, the peripheral zone of the host-cell cytoplasm appears normal. Inside the peripheral region of the 0.4–1.0 mm xenoma, the host-cell component largely disintegrates in the presence of microsporidan parasites undergoing sporogenesis.     

Intracellular Development of the Microsporidan Glugea anomala Moniez in Hypertrophying Migratory Cells of the Fish Gasterosteus aculeatus L., an Example of the Formation of "Xenoma" Tumors

SYNOPSIS. The conclusion drawn in 1921 that the large nuclei in the cytoplasmic cortex of Glugea cysts are not vegetative nuclei of the microsporidan but nuclei of the hypertrophied host cell was based on the discovery of early developmental stages in the mesenchyme of stickleback larvae experimentally fed Glugea spores. This observation had been made on serial sections from experiments done in 1912. The intracellular development of the microsporidan could be followed up in this material only thru the 1st stages of schizogony. Renewed infection experiments, done still in 1921 on a much broader basis, have fully confirmed the previous findings, as briefly stated in 1922. On this material, the intracellular development of G. anomala has been followed up in recent years from uninucleate host cells 7 μ in diameter, interpreted as wandering cells in the mesenchyme, until they became macroscopic multinucleate cysts, in which schizogony and sporogony of the microsporidan produced innumerable vegetative stages and spores of Glugea. The details of the developmental processes are described in the present paper.
The multinucleate host cell and the intracellular parasites together form one of the symbiotic complexes for which the term "xenom" or "xenoma" has been used by me since 1949. By a sequence of amitotic nuclear divisions, the uninucleate host cell in the Glugea xenomas of Gasterosteus becomes plurinucleate in contrast to the usual structure of other xenomas of fish.
Already in 1921, I thought that the host cell in the Glugea xenomas may have phagocytic properties. The observation of accumulation of granules from pigment cells in some of the Glugea xenomas has now verified this supposition.     

A new microsporidian parasite, Potaspora morhaphis n. gen., n. sp. (Microsporidia) infecting the Teleostean fish, Potamorhaphis guianensis from the River Amazon. Morphological, ultrastructural and molecular characterization

A fish-infecting Microsporidia Potaspora morhaphis n. gen., n. sp. found adherent to the wall of the coelomic cavity of the freshwater fish, Potamorhaphis guianensis, from lower Amazon River is described, based on light microscope and ultrastructural characteristics. This microsporidian forms whitish xenomas distinguished by the numerous filiform and anastomosed microvilli. The xenoma was completely filled by several developmental stages. In all of these stages, the nuclei are monokaryotic and develop in direct contact with host cell cytoplasm. The merogonial plasmodium divides by binary fission and the disporoblastic pyriform spores of sporont origin measure 2.8+/-0.3 x 1.5+/-0.2 microm. In mature spores the polar filament was arranged into 9-10 coils in 2 layers. The polaroplast had 2 distinct regions around the manubrium and an electron-dense globule was observed. The small subunit, intergenic space and partial large subunit rRNA gene were sequenced and maximum parsimony analysis placed the microsporidian described here in the clade that includes the genera Kabatana, Microgemma, Spraguea and Tetramicra. The ultrastructural morphology of the xenoma, and the developmental stages including the spores of this microsporidian parasite, as well as the phylogenetic analysis, suggest the erection of a new genus and species.     

Glugea vincentiae n. sp. (Microsporidia: Glugeidae) infecting the Australian marine fish Vincentia conspersa (Teleostei: Apogonidae)

A parasite of the marine fish Vincentia conspersa was examined by light microscopy and transmission electron microscopy. This parasite develops in the subcutaneous tissue of the body and fins, forming spherical xenomas about 1-2 mm in diameter surrounded by a layer of amorphous material. The observed characteristics of the new parasite are in line with those of the other Glugea species; merogony takes place in the outer zone of the cytoplasm of the host cell, sporogony takes place in sporophorous vesicles, and mature spores are located in the central part of the xenoma. Meronts were cylindrical uninucleate or occasionally triradiate multinucleate, with plasmodia in direct contact with the host cytoplasm. Sporogonic plasmodia divided by multiple cleavage to produce sporoblast mother cells, which after binary fission became sporoblasts. Two types of spores were recognized, both uninucleate, i.e., ovoid or slightly ovoid microspores with a mean size of 5.1 x 2.2 microm and much less frequent as elongated oval macrospores with a mean size of 8.9 x 3.1 microm. The polar tube has between 12 and 14 coils arranged in 1, 2, or 3 layers. Taken together, these characteristics suggest that this microsporidian infecting V. conspersa is a new species of Glugea, which we have named Glugea vincentiae.     

Ultrastructural study of the late stages of Loma salmonae development in the gills of experimentally infected rainbow trout

The main objective of this investigation was to examine the ultrastructural features of gills from rainbow trout experimentally infected with Loma salmonae to determine the morphological events that occur during the late stages of development of this parasite. Peripheral distribution of the mature parasites inside round xenomas was observed at weeks 5 and 6 postexposure (PE), but eventually the parasite occupied the entire xenoma. Degenerative changes were observed only in immature parasites at week 7 PE, and eventually an inflammatory reaction with a cellular infiltration was directed against mature spores. Round, flattened, and irregular shaped xenomas were observed at week 8 PE. The round xenomas showed a severe inflammatory response with disintegration of the xenoma membrane. This event was accompanied by eversion of polar tubes within the attacked xenoma and by the simultaneous presence of 2 tubular appendages, the type I and II tubules. Flattened xenomas were observed below the endothelium of gill lamella arteries. The irregular xenomas were located in the connective tissue of the gill filament and showed multiple projections occupied by spores. Both flattened and irregular xenomas showed no evidence of inflammatory reaction. An earlier proposed hypothesis is expanded to explain how L. salmonae is implanted beneath lamellar endothelium and within filament connective tissue.     

The diversity of microsporidia in parasitic copepods (Caligidae: Siphonostomatoida) in the Northeast Pacific Ocean with description of Facilispora margolisi n. g., n. sp. and a new Family Facilisporidae n. fam

Three distinct microsporidia were identified from parasitic copepods in the northeast Pacific Ocean. Sequencing and phylogenetic analysis of a partial small subunit ribosomal RNA gene (SSU rDNA) sequence identified a genetically distinct variety of Desmozoon lepeophtherii from Lepeophtheirus salmonis on cultured Atlantic salmon Salmo salar, and this was confirmed by transmission electron microscopy. Phylogenetic analysis resolved the SSU rDNA sequence of the second organism in a unique lineage that was most similar to microsporidia from marine and brackish water crustaceans. The second occurred in L. salmonis on Atlantic, sockeye Oncorhynchus nerka, chum O. keta and coho O. kisutch salmon, in Lepeophtheirus cuneifer on Atlantic salmon, and in Lepeophtheirus parviventris on Irish Lord Hemilepidotus hemilepidotus. Replication occurred by binary fission during merogony and sporogony, diplokarya were not present, and all stages were in contact with host cell cytoplasm. This parasite was identified as Facilispora margolisi n. g., n. sp. and accommodated within a new family, the Facilisporidae n. fam. The third, from Lepeophtheirus hospitalis on starry flounder Platichthys stellatus, was recognized only from its unique, but clearly microsporidian SSU rDNA sequence. Phylogenetic analysis placed this organism within the clade of microsporidia from crustaceans.     

Three-dimensional electron microscopy analysis reveals endopolygeny-like nuclear architecture segregation in Plasmodium oocyst development

The genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the production of haploid parasites in the Plasmodium life cycle: the oocyst stage in mosquitoes and the liver and blood stages in mammalian hosts. The Plasmodium oocyst stage plays an important role in the production of the first generation of haploid parasites. Nuclear division is the most important event that occurs during the proliferation of all eukaryotes. However, obtaining the details of nuclear division at the oocyst stage is challenging owing to difficulties in preparation. In this study, we used focused-ion-beam-milling combined with scanning-electron-microscopy to report the 3D architecture during nuclear segregations in oocyst stage. This advanced technology allowed us to analyse the 3D details of organelle segregation inside the oocyst during sporogony formation. It was revealed that multiple nuclei were involved with several centrosomes in one germ nucleus during sporozoite budding (endopolygeny). Our high-resolution 3D analysis uncovered the endopolygeny-like nuclear architecture of Plasmodium in the definitive host. This nuclear segregation was different from that in the blood stage, and its similarity to other apicomplexan parasite nuclear divisions such as Sarcocystis is discussed.     

Ultrastructural evidence of autoinfection in the gills of Atlantic cod Gadus morhua infected with Loma sp. (phylum Microsporidia)

Infection by a microsporidian of the genus Loma was found in gills of cod Gadus morhua. Xenomas contained parasites in multiple stages of development. Some spores looked empty and had everted polar tubes, which were either straight or coiled. These polar tubes were scattered throughout the xenoma cytoplasm, and some of them pierced the plasma membrane. Those outside of the xenoma penetrated neighboring cells, including blood cells. These observations suggest that a mechanism of autoinfection could occur in blood cells and gill tissue, perpetuating the disease in the host.     

Salivary gland of the tick vector of east coast fever. IV. Cell type selectivity and host cell responses to Theileria parva

Responses of cells in the tick salivary gland to parasitism by Theileria parva were studied by electron microscopy. The gland is composed of three distinct types of acini (I, II, III) which together include ten or more different cell types. Of some 30 infected cells observed in the present study, all were E-cells of acinus III. The parasite thus exhibits a high degree of selectivity for acinus and cell type. The glandular cell invaded undergoes massive hypertrophy and accumulates glycogen deposits in its cytoplasm which may serve as an energy source for the growing intracellular parasite. As synthesis of its secretory material declines the product is packaged in progressively smaller secretory granules. The extensive arrays of endoplasmic reticulum are dismantled and eliminated in autophagic vacuoles. Excess secretory granules are also broken down by crinophagy. After 4 days, sporogony is completed and the host cell contains 30,000–50,000 sporozoites in an electron-lucent cytoplasm largely devoid of cytomembranes and secretory granules. Mitochondria are still present and normal in appearance. The loss of basophilia and secretory granules observed heretofore by light microscopy have been attributed to ingestion and destruction of host organelles by the parasite. The pallid appearance of the cytoplasm has been interpreted as a sign of impending degeneration of the host cell. In electron micrographs no ingestion of organelles by the parasite or degenerative changes were found. The host cell clearly remains viable and metabolically active throughout sporogony. The striking changes in its ultrastructure result from active elimination of organelles and inclusions by the host cell itself in response to parasitism.     

Effects of dexamethasone on host innate and adaptive immune responses and parasite development in rainbow trout Oncorhynchus mykiss infected with Loma salmonae

The effects of dexamethasone (dex) treatment on infections with the microsporidian parasite, Loma salmonae and the effects of dex on initiation of the adaptive immune response were investigated in rainbow trout, Oncorhynchus mykiss experimentally infected with the parasite. Dex treatment resulted in significantly higher infections with the parasite in the gills and other internal organs, suggesting that dex inhibits aspects of the innate immune response to L. salmonae; the heavier infections in the gills and organs of rainbow trout resembled infections seen in Chinook salmon. Mean xenoma counts per microscope field in the gills of fish infected with L. salmonae treated with dex or left untreated were 169 and 30, respectively. Although higher numbers of xenomas were observed in dex treated fish, the xenomas were generally smaller in size than in infected control fish. The xenomas in dex treated fish showed morphological signs of degeneration including loss and degeneration of early parasite stages, accumulation of amorphous material in xenomas, and infiltration with phagocytic cells containing degenerated parasites. The xenomas in infected untreated fish had larger xenomas with a more uniform size and contained identifiable parasite stages in the cytoplasm. According to this study, once fish have developed an adaptive immune response to the parasite by previous exposure, then fish have 100% protection to reinfection even when treated with heavy doses of dex. L. salmonae immune fish treated or untreated with dex during reinfection with the parasite developed no xenomas in the gills 6 weeks post reinfection. These results indicate that once the cellular response is primed to L. salmonae, then dex related immunosuppression does not reduce the effectiveness of the adaptive immune response.     

Microgemma vivaresi n. sp. (Microsporidia, Tetramicridae), infecting liver and skeletal muscle of sea scorpions, Taurulus bubalis (Euphrasen 1786) (Osteichthyes, Cottidae), an inshore, littoral fish

The ultrastructure of a new microsporidian species Microgemma vivaresi n. sp. causing liver cell xenoma formation in sea scorpions, Taurulus bubalis, is described. Stages of merogony, sporogony, and sporogenesis are mixed in the central cytoplasm of developing xenomas. All stages have unpaired nuclei. Uninucleate and multinucleate meronts lie within vacuoles formed from host endoplasmic reticulum and divide by binary or multiple fission. Sporonts, no longer in vacuoles, deposit plaques of surface coat on the plasma membrane that cause the surface to pucker. Division occurs at the puckered stage into sporoblast mother cells, on which plaques join up to complete the surface coat. A final binary fission gives rise to sporoblasts. A dense globule, thought to be involved in polar tube synthesis, is gradually dispersed during spore maturation. Spores are broadly ovoid, have a large posterior vacuole, and measure 3.6 microm x 2.1 microm (fresh). The polar tube has a short wide anterior section that constricts abruptly, then runs posteriad to coil about eight times around the posterior vacuole with granular contents. The polaroplast has up to 40 membranes arranged in pairs mostly attached to the wide region of the polar tube and directed posteriorly around a cytoplasm of a coarsely granular appearance. The species is placed alongside the type species Microgemma hepaticusRalphs and Matthews 1986 within the family Tetramicridae, which is transferred from the class Dihaplophasea to the class Haplophasea, as there is no evidence for the occurrence of a diplokaryotic phase.     

Paranucleospora theridion n. gen., n. sp. (Microsporidia,Enterocytozoonidae) with a Life Cycle in the Salmon Louse (Lepeophtheirus salmonis,Copepoda) and Atlantic Salmon (Salmo salar)

ABSTRACT. Paranucleospora theridion n. gen, n. sp., infecting both Atlantic salmon (Salmo salar) and its copepod parasite Lepeophtheirus salmonis is described. The microsporidian exhibits nuclei in diplokaryotic arrangement during all known life‐cycle stages in salmon, but only in the merogonal stages and early sporogonal stage in salmon lice. All developmental stages of P. theridion are in direct contact with the host cell cytoplasm or nucleoplasm. In salmon, two developmental cycles were observed, producing spores in the cytoplasm of phagocytes or epidermal cells (Cycle‐I) and in the nuclei of epidermal cells (Cycle‐II), respectively. Cycle‐I spores are small and thin walled with a short polar tube, and are believed to be autoinfective. The larger oval intranuclear Cycle‐II spores have a thick endospore and a longer polar tube, and are probably responsible for transmission from salmon to L. salmonis. Parasite development in the salmon louse occurs in several different cell types that may be extremely hypertrophied due to P. theridion proliferation. Diplokaryotic merogony precedes monokaryotic sporogony. The rounded spores produced are comparable to the intranuclear spores in the salmon in most aspects, and likely transmit the infection to salmon. Phylogenetic analysis of P. theridion partial rDNA sequences place the parasite in a position between Nucleospora salmonis and Enterocytozoon bieneusi. Based on characteristics of the morphology, unique development involving a vertebrate fish as well as a crustacean ectoparasite host, and the results of the phylogenetic analyses it is suggested that P. theridion should be given status as a new species in a new genus.     

Population dynamics of sporogony for Plasmodium vivax parasites from western Thailand developing within three species of colonized Anopheles mosquitoes

Background

The population dynamics of Plasmodium sporogony within mosquitoes consists of an early phase where parasite abundance decreases during the transition from gametocyte to oocyst, an intermediate phase where parasite abundance remains static as oocysts, and a later phase where parasite abundance increases during the release of progeny sporozoites from oocysts. Sporogonic development is complete when sporozoites invade the mosquito salivary glands. The dynamics and efficiency of this developmental sequence were determined in laboratory strains of Anopheles dirus, Anopheles minimus and Anopheles sawadwongporni mosquitoes for Plasmodium vivax parasites circulating naturally in western Thailand.

Methods

Mosquitoes were fed blood from 20 symptomatic Thai adults via membrane feeders. Absolute densities were estimated for macrogametocytes, round stages (= female gametes/zygotes), ookinetes, oocysts, haemolymph sporozoites and salivary gland sporozoites. From these census data, five aspects of population dynamics were analysed; 1) changes in life-stage prevalence during early sporogony, 2) kinetics of life-stage formation, 3) efficiency of life-stage transitions, 4) density relationships between successive life-stages, and 5) parasite aggregation patterns.

Results

There was no difference among the three mosquito species tested in total losses incurred by P. vivax populations during early sporogony. Averaged across all infections, parasite populations incurred a 68-fold loss in abundance, with losses of ca. 19-fold, 2-fold and 2-fold at the first (= gametogenesis/fertilization), second (= round stage transformation), and third (= ookinete migration) life-stage transitions, respectively. However, total losses varied widely among infections, ranging from 6-fold to over 2,000-fold loss. Losses during gametogenesis/fertilization accounted for most of this variability, indicating that gametocytes originating from some volunteers were more fertile than those from other volunteers. Although reasons for such variability were not determined, gametocyte fertility was not correlated with blood haematocrit, asexual parasitaemia, gametocyte density or gametocyte sex ratio. Round stages and ookinetes were present in mosquito midguts for up to 48 hours and development was asynchronous. Parasite losses during fertilization and round stage differentiation were more influenced by factors intrinsic to the parasite and/or factors in the blood, whereas ookinete losses were more strongly influenced by mosquito factors. Oocysts released sporozoites on days 12 to 14, but even by day 22 many oocysts were still present on the midgut. The per capita production was estimated to be approximately 500 sporozoites per oocyst and approximately 75% of the sporozoites released into the haemocoel successfully invaded the salivary glands.

Conclusion

The major developmental bottleneck in early sporogony occurred during the transition from macrogametocyte to round stage. Sporozoite invasion into the salivary glands was very efficient. Information on the natural population dynamics of sporogony within malaria-endemic areas may benefit intervention strategies that target early sporogony (e.g., transmission blocking vaccines, transgenic mosquitoes).     

Calcium and Hydrogen Ion Concentrations in the Parasitophorous Vacuoles of Epithelial Cells Infected with the Microsporidian Encephalitozoon hellem

ABSTRACT. Microsporidia of the genus Encephalitozoon undergo merogony and sporogony in a parasitophorous vacuole within the host cell. Cultured green monkey kidney cells infected with Encephalitozoon hellem were loaded with the fluorescent dyes fura-2 or BCECF in order to measure intracellular concentrations of calcium and hydrogen ions respectively. Both the parasitophorous vacuole calcium concentration and pH values resembled those of the host cell cytoplasm in infected cells. Calcein entered the parasitophorous vacuole but not other host cell vacuoles or parasite stages within the parasitophorous vacuole. The lack of a pH or calcium concentration gradient across the parasitophorous vacuole membrane and the permeability of this membrane to a large anion such as calcein suggest that the vacuole membrane surrounding E. hellem resembles that surrounding some other intracellular parasites such as Toxoplasma gondii. A potential role is discussed for the parasitophorous vacuole calcium concentration in germination in situ.     

THE ROLE OF SECONDARY PIT CONNECTIONS IN RED ALGAL PARASITISM1

Secondary pit connections are common between cells of hosts and parasites in the widespread phenomenon of red algal parasitism. The DNA-specific fluorochrome 4′,-6-diamidino-2-phenylindole (DAPI) reveals that in host-parasite secondary pit connection (SPC) formation between the parasitic red alga Choreocolax polysiphoniae and its host Polysiphonia confusa, a nucleus and other cytoplasmic components of the parasite are delivered into the cytoplasm of a host cell. Host cells receive large numbers of parasite nuclei and these, apparently arrested in G¹, are maintained intact in host cells for periods of several weeks. Within these enlarged, differentiated cells, starch accumulates and cytoplasmic organelles proliferate as the central vacuole decreases in size. Host nuclear DNA synthesis is stimulated in the infected host cell, resulting in an increase in the number of host nuclei, or an increase in DNA in each of the existing host nuclei (i.e. somatic polyploidy). Occasionally, infected host cells will recommence division and engender a new host branch. Microspectrofluorometry of nuclear DNA quantitatively confirms not only the identity and transfer of parasite nuclei to host cells, but also the transfer of parasite nuclei to other parasite cells. Measurements also reveal that the single nucleus of Choreocolax becomes progressively more polyploid as cells become larger and more highly differentiated. Secondary pit connection formation between Choreocolax and Polysiphonia provides the mechanism for the transfer of parasite genetic information (via the parasite nucleus and cytoplasm) into the host. The parasite nuclei may thereby control and redirect the physiology of the host for the benefit of the parasite.     

Cryo transmission X-ray imaging of the malaria parasite,P. falciparum

Cryo transmission X-ray microscopy in the “water window” of photon energies has recently been introduced as a method that exploits the natural contrast of biological samples. We have used cryo tomographic X-ray imaging of the intra-erythrocytic malaria parasite, Plasmodium falciparum, to undertake a survey of the cellular features of this important human pathogen. We examined whole hydrated cells at different stages of growth and defined some of the structures with different X-ray density, including the parasite nucleus, cytoplasm, digestive vacuole and the hemoglobin degradation product, hemozoin. As the parasite develops from an early cup-shaped morphology to a more rounded shape, puncta of hemozoin are formed; these coalesce in the mature trophozoite into a central compartment. In some trophozoite stage parasites we observed invaginations of the parasite surface and, using a selective permeabilization process, showed that these remain connected to the RBC cytoplasm. Some of these invaginations have large openings consistent with phagocytic structures and we observed independent endocytic vesicles in the parasite cytoplasm which appear to play a role in hemoglobin uptake. In schizont stage parasites staggered mitosis was observed and X-ray-dense lipid-rich structures were evident at their apical ends of the developing daughter cells. Treatment of parasites with the antimalarial drug artemisinin appears to affect parasite development and their ability to produce the hemoglobin breakdown product, hemozoin.     

Ultrastructural details of the xenoma of Loma myrophis (phylum Microsporidia) and extrusion of the polar tube during autoinfection

Xenomas of the recently described new microsporidian species Loma myrophis parasitizing the gut tissue of the Amazonian fish Myrophis platyrhynchus (family Ophichthidae) were described by light- and transmission-electron microscopy. The xenoma consisted of a thin fibrillar wall that surrounded a hypertrophic host cell cytoplasm containing numerous microsporidian developmental stages and spores. Several spores showed different stages of natural extrusion of the polar tube. Numerous longitudinal and transverse sections of the extruded polar tubes were observed in developing life-cycle stages (spores excepted), the nucleus of hypertrophic host cell, the xenoma wall and surrounding fibroblasts. The extruded polar tubes were projected in all directions with no preferential orientation. These aspects suggested that autoinfection occurred within this xenoma.