Pathogenesis of Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) in susceptible noctuid larvae

Helicosporidium sp. is a unique, achlorophyllous green alga that has been reported to infect various insect orders, including Lepidoptera, Diptera, and Coleoptera. The infectious cyst stage is ingested by the host, ruptures in the midgut lumen, and releases a filamentous cell. Histopathological examinations using larvae of a susceptible noctuid host, Spodoptera exigua, showed both cysts and filamentous cells affiliated with the microvillar lining of the midgut epithelium. A considerable proportion of the ingested cysts (22-39%) were recovered in feces collected 24 h after ingestion. A small number of filamentous cells passed the midgut epithelium and entered the hemocoel within 4-24 h after cyst ingestion. After 48 h, vegetative cell stages were detected in the hemolymph, followed by a 4- to 5-day period of increasing multiplication. Cyst differentiation in the colonized hemolymph began 6-7 days after the treatment.     

Development of the insect pathogenic alga Helicosporidium

This study examined the morphogenesis and replication dynamics of the different life stages (cysts, filamentous cells, vegetative cells) of Helicosporidium sp., a non-photosynthetic, entomopathogenic alga. The isolate (SjHe) used originated from an infected black fly larva. Filamentous cell transformation into vegetative cells and autosporulation during vegetative cell replication were observed under controlled in vitro conditions. The transformation process was initiated by a partial swelling of the filamentous cell along with the reorganization of the nuclear material. Two subsequent nuclear and cell divisions resulted in the release of 4 rod-shaped daughter cells, which divided into oval to spherical vegetative cells. These underwent several cycles of autosporogenic cell division. Multiple-passaged vegetative cell cultures formed non-motile, adherent cell clusters (palmelloid colonies). Vegetative replication dynamics were also observed in 2 experimental noctuid hosts, Spodoptera exigua and Helicoverpa zea. The average density of helicosporidial cells produced per microliter hemolymph exceeded cell concentrations obtained in vitro by 15- and 46-fold in S. exigua and H. zea, respectively. Cyst morphogenesis was only observed in the hemolymph, whereas no cysts differentiated at various in vitro conditions.     

Detection of Helicosporidium spp. in metagenomic DNA

Distinct isolates of the invertebrate pathogenic alga Helicosporidium sp., collected from different insect hosts and different geographic locations, were processed to sequence the 18S rDNA and β-tubulin genes. The sequences were analyzed to assess genetic variation within the genus Helicosporidium and to design Helicosporidium-specific 18S rDNA primers. The specificity of these primers was demonstrated by testing not only on the Helicosporidium sp. isolates, but also on two trebouxiophyte algae known to be close Helicosporidium relatives, Prototheca wickerhamii and Prototheca zopfii. The genus-specific primers were used to develop a culture-independent assay aimed at detecting the presence of Helicosporidium spp. in environmental waters. The assay was based on the PCR amplification of 18SrDNA gene fragments from metagenomic DNA preparations, and it resulted in the amplification of detectable products for all sampled sites. Phylogenetic analyses that included the environmental sequences demonstrated that all amplification products clustered in a strongly supported, monophyletic Helicosporidium clade, thereby validating the metagenomic approach and the taxonomic origin of the produced environmental sequences. In addition, the phylogenetic analyses established that Helicosporidium spp. isolated from coleopteran hosts are more closely related to each other than they are to the isolate collected from a dipteran host. Finally, the phylogenetic trees depicted intergeneric relationships that supported a Helicosporidium-Prototheca cluster but did not support a Helicosporidium-Coccomyxa grouping, suggesting that pathogenicity to invertebrates evolved at least twice independently within the trebouxiophyte green algae.     

The non-photosynthetic, pathogenic green alga Helicosporidium sp. has retained a modified, functional plastid genome

A fragment of the Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) plastid genome has been sequenced. The genome architecture was compared to that of both a non-photosynthetic relative (Prototheca wickerhamii) and a photosynthetic relative (Chlorella vulgaris). Comparative genomic analysis indicated that Helicosporidium and Prototheca are closely related genera. The analyses also revealed that the Helicosporidium sp. plastid genome has been rearranged. In particular, two ribosomal protein-encoding genes (rpl19 and rps23) appeared to have been transposed, or lost from the Helicosporidium sp. plastid genome. RT-PCR reactions demonstrated that the retained plastid genes were transcribed, suggesting that, despite rearrangement(s), the Helicosporidium sp. plastid genome has remained functional. The modified plastid genome architecture is a novel apomorphy that indicates that the Helicosporidia are highly derived green algae, more so than Prototheca spp. As such, they represent a promising model to study organellar genome reorganizations in parasitic protists.     

Transitions in trophectoderm cellular shape and cytoskeletal organization in the elongating pig blastocyst

Changes in cellular shape and filamentous actin (f-actin) organization within the trophectoderm of pig embryos have been studied by fluorescence microscopy during the transitions from spherical to filamentous blastocysts. Cells comprising the trophectoderm of spherical, ovoid, tubular, and filamentous blastocysts are distinctive in their shape, size, and organization of membrane-associated f-actin. Trophectodermal cells of spherical and ovoid embryos are both generally circular in shape. However, as the spherical embryo acquires an ovoid shape, uniformally distributed apical cell surface microvilli relocate to the apical intercellular margins of adjoining trophectodermal cells. Transitional modifications in cellular shape and f-actin organization are observed in tubular blastocysts when apical cell surface microvilli reappear. In elongating filamentous blastocysts, trophectodermal cells assume a spindle-shaped morphology. The f-actin associated with the apical surface is diminished whereas the associated with the basolateral membrane predominates, especially in constricted regions of the blastocyst. These observations, in conjunction with morphometric parameters of trophectodermal cells and whole blastocysts, are discussed in relation to the role of the actin cytoskeleton in processes that modify trophectodermal cell shape and function in the elongating pig blastocyst.     

Helicosporidium parasiticum Keilin Infection in the Caterpillar of a Hepialid Moth in Argentina

SYNOPSIS A case of infection by Helicosporidium parasiticum Keilin, 1921, in a hepialid caterpillar from Argentina is described. The pathogen produces deep proliferation starting from a cuticular callus, with numerous cysts occluded by multiple fibrous layers of connective tissue. Other cysts encapsulated in layers of lymphocytes and cysts in the lobes of the fat body are present in different parts of the body. Centers of melanization occur in most cysts. The pathway of infection, the reaction of the host and some structural features of the parasite are typical for fungus infections. Transfer of the genus Helicosporidium from Protozoa to primitive Ascomycetes, Saccharomycetaceae, is proposed.     

Helicosporidium infection of the great European spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

An infection of the great European spruce bark beetle Dendroctonus micans (from Turkey) by the parasitic green alga Helicosporidium is described. This is the first time that Helicosporidium has been found to infect a bark beetle (Scolytidae) and the first time that a naturally infected beetle has been reported for the Eurasian continent. The typical cysts of Helicosporidium contain three ovoid cells and one helical, filamentous cell. The morphological characteristics are revealed by light and electron microscopy. Distinct electron-dense inclusions with a peculiar ultrastructure may represent pyrenoids. Since D. micans is an important pest, the discovery of a natural pathogen may offer a chance for biological control.     

Cultivation and Lyophilization of Mastigina sp.

SYNOPSIS Mastigina sp. is an amoeboid flagellate isolated from pine frass collected in the Guadarrama Mountains in Spain. It feeds on bacteria and yeasts. It prefers yeasts that produce extracellular polysaccharides, and the 2 species that have been used predominantly for cultivation of the flagellate are Pachysolen tannophilus and Hansenula holstii. Mastigina sp. is easily isolated in axenic culture and grows abundantly therein. Its locomotive form, averaging 27 μ in length, resembles that of a limax amoeba, with a vesiculate nucleus at the anterior end. Cells are capable of simultaneous movement by pseudopodia and flagella. It develops rapidly on dead or living yeast cells in shaken cultures and the trophozoites may convert quantitatively to cysts. The cysts remain viable for long periods of time in refrigerated suspensions and in the lyophilized state. They are spherical or ovoid and smooth-walled cysts; the trophozoite emerges from them by breaking the wall.     

New species of Eimeria (Apicomplexa: Eimeriidae) from the domesticated goat Capra hircus in New Zealand

Three new species of the genus Eimeria Schneider, 1875 are described from the faeces of domesticated goats in New Zealand. Oöcysts of E. capralis n. sp. are ellipsoidal, 29.2 × 19.7 (25–34 × 17–24) μm, with a distinct micropylar cap. The sporocysts are broadly ovoid, the Stieda body is present and the sporocyst residuum consists of many scattered granules. Sporozoites lie lengthwise head to tail in the sporocyst. Oöcysts of E. masseyensis n. sp. are broadly ellipsoid to ovoid, 22.3 × 17.4 (18–25 × 15–19) μm, with a distinct micropylar cap. The polar granules are shattered into fine granules, the sporocysts are elongate ovoid and the Stieda body is present. Oöcysts of E. charlestoni n. sp. are ellipsoidal, 22.9 × 17.4 (20–25 × 16–19) μm, with no micropylar cap. Its oöcysts are distinctive, with elongate sporocysts containing very prominent refractile bodies.     

The endogenous development of two new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from Thai geckoes

Two new species of Isospora are described from Thai geckoes. I. platyurusi n. sp. in Cosymbotus platyurus from Chiang Mai and Khon Kaen and I. uptoni n. sp. in Hemidactylus frenatus from Chiang Mai. Oöcysts of I. platyurusi are spherical to subspherical, 19–22.5 × 16.5–21.5 μm and have two ovoid sporocysts of 11.5–14 × 7.5–9 μm. Oöcysts of I. uptoni are spherical to subspherical, 24–31.5 × 20–27.5 μm and have two ovoid sporocysts of 12.5–15 × 9–11.5 μm. Both species have sporocysts with Stieda bodies and undergo endogenous development in the nucleus of the host gut epithelial cells. On the completion of merogony and gamogony, the host nucleus is reduced to a thin envelope.     

Nuclear morphometric features of epithelial cells lining keratocysts

OBJECTIVE: To investigate the nuclear morphometric features of epithelial cells lining keratocysts and some other odontogenic cysts. STUDY DESIGN: All cases were selected from the archives of the Department of Pathology, Gülhane Military Medical Academy, as follows: 20 keratocysts and 10 dentigerous and 10 radicular cysts. Nuclear morphometric variables were measured on hematoxylin and eosin-stained histologic slides. Basal and intermediate cells of the epithelium were evaluated separately. Nuclei of the cells were outlined interactively and measured using a specially written macro program. Area, feret ratio (ratio of the longest nuclear axis to the shortest one) and circularity (F circle) of the nuclei were calculated. Additionally, nuclear densitometric analysis was performed on the keratocyst cases. RESULTS: The number of cells in the basal layer (cell density) was higher in keratocysts than in other cysts. The mean nuclear area of basal cells was smaller than of intermediate cells in both keratocysts and other cysts (P < .001). The feret ratio values revealed that basal cell nuclei of keratocysts were more ovoid as compared to those of other cysts (P < .001). Nuclear densitometric findings showed that the DNA indices of all keratocyst cases were close to 1.0, and the cells were considered diploid. CONCLUSION: Increased cell density, a more ovoid nuclear shape and more variation in the size of basal layer cell nuclei in keratocysts were helpful in differentiating these lesions from other odontogenic cysts.     

The peculiarities of Blastocrithidia triatomae

Blastocrithidia triatomae parasitizes triatomine bugs, the vectors of Chagas disease. Co-cultivation with a host-derived cell line permits continuous culture but the host cells are destroyed. Removal of the reduviid cells induces the formation of drought-resistant cysts, but the factors that induce encystment are unknown. Excystment is triggered after the onset of blood digestion in the insect host, a transition that is associated with unusual ultrastructural alterations. Günter Schaub, Dagmar Reduth and Mary Pudney believe that B. triatomae is a good candidate for the biological control of Chagas disease, not least because of its capacity to form highly resistant cysts in vitro.     

Ultrastructural and biochemical characterization of promastigote and cystic forms of Leptomonas wallacei n. sp. isolated from the intestine of its natural host Oncopeltus fasciatus (Hemiptera: Lygaeidae)

Promastigote forms of a trypanosomatid were isolated from the third and fourth ventricles of the midgut and from the hindgut of the phytophagous hemipteran Oncopeltus fasciatus. Some individuals had adhered to its anterior region, close to the flagellar pocket, or to the flagellum up to four rounded aflagellated forms known as straphangers cysts. Scanning electron microscopy revealed that the flagellated forms presented a twisted cell body and a long flagellum, and the cysts, smaller than the parental promastigote, had a nascent flagellum. Transmission electron microscopy showed that promastigotes were typical, while cystic forms were ovoid dense cells devoid of a cyst wall, but presenting a cell coat, a special subpellicular region limited by a membrane unit, and a condensed cytoplasm. The kinetoplast-DNA fibrils appeared as dense spots and the condensed chromatin was arranged in a labyrinthic structure. Desmosome-like structures, observed in the region of adhesion of the precystic forms to the parental promastigote, could explain how cysts remain attached to the mother cell during the encystation process. Release of membranes from the surface of promastigotes and cysts seems to be correlated with the condensation of the cytoplasm during encystment. Morphological and isozyme analyses indicated that this trypanosomatid belongs to the genus Leptomonas. The molecular karyotype of this isolate was compared with that of a strain of Leptomonas oncopelti obtained from Oncopeltus varicolor by contour-clamped homogeneous electric field (CHEF) electrophoresis and revealed similar DNA banding patterns between 2,200-825 Kb, but not in lower bands (825-225 Kb). This suggested that the isolate from O. fasciatus and that from O. varicolor were not identical. Based on our findings we are describing Leptomonas wallacei n. sp. for our isolate from O. fasciatus.     

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.     

Biotechnological implications of filamentation in Saccharomyces cerevisiae

The genetics governing the morphological switch from round or ovoid cells to filamentous growth in Saccharomyces cerevisiae has received significant interest in relation to sensing and signaling pathways as well as the control of cell processes including budding, elongation and adhesion. Little is known about the environmental signals which trigger these morphological changes from a biotechnological point of view. This review aims to highlight the main causes of filamentous growth in S. cerevisiae in its industrial setting with the purpose of stimulating additional studies within this field.     

Sarcocystis Infection in Wild Reindeer (Rangifer Tarandus) From Hardangervidda in Southern Norway: with a Description of The Cysts of Sarcocystis Hardangeri N. Sp.

Fresh preparations of micro-isolated sarcosysts from skeletal muscle of 5 wild reindeer were examined by light microscopy. Slender, spindelshaped cysts measuring 821 × 60 µm, and having short knob-like cyst wall protrusions were found in all animals. In 1 animal cysts different in structure from the cysts of the 4 previously known Sarcocystis spp. of reindeer were found, These cysts are considered to be cysts of a new Sarcocystis sp. of reindeer, for which the name Sarcocystis hardangeri has been proposed. S. hardangeri n. sp. had macroscopic, ovoid to cylindrical cysts measuring 1667 (900–2570) × 819 (450–1575) µm. The cysts were surrounded by a 8–10 µm thick layer of fibrillar material. After removal of this layer, relatively few and irregularly spaced, slanting protrusions became visible. The 20–30 µm long protrusions were tongue-like, and were lying close to the surface of the cyst. Cysts of S. grueneri, S. rangiferi and S. tarandi were not demonstrated in the 5 wild reindeer examined.     

Nuclear bodies in the normal and hyperfunctional human adrenal cortex

Adrenal pieces obtained from six female patients, three without increased adrenocortical function and three with Cushing's disease, showed, in all adrenal cortex zones, cells containing simple and complex nuclear bodies. The simple nuclear bodies were spherical or ovoid and had a filamentous structure surrounded by a clear halo. Complex nuclear bodies were more numerous and heterogeneous in patients with adrenal pathology, and they were spherical with a proteinaceous filamentous capsule surrounding a core; the core was granular, filamentous or a mixture of granular and filamentous material, sometimes with a reticular or concentric arrangement. Some bodies showed vacuolar or multilocular aspect, and others had a close relationship with the nucleolus or appeared near the interchromatin granules. The meaning of adrenal nuclear bodies is discussed as well as their relationship with ACTH stimulation.