Diplokaryotic Nuclei Research Articles

Septin homologs cooperating in the Proliferative Stage of Microsporidia Nosema bombycis

The single-celled pathogen Nosema bombycis, that can infect silkworm Bombyx mori and other lepidoptera including Spodoptera, is the first identified Microsporidia which has diplokaryotic nuclei throughout the life cycle. Septin proteins can form highly ordered filaments, bundles or ring structures related to the cytokinesis in fungi. Here, three septin proteins (NbSeptin1, NbSeptin2 and NbSeptin3) from Nosema bombycis CQ I are described. These proteins, appear to be conserved within the phylum Microsporidia. NbSeptins transcripts were detected throughout the pathogen developmental cycle and were significantly enhanced from second days of infection, which lead to our hypothesis that NbSeptins play a role in merogony. Immunofluorescence assay (IFA) revealed a broad distribution of NbSeptins in meronts and partly co-localization of NbSeptins. Interestingly, in some of meronts, NbSeptin2 and NbSeptin3 showed localization between the nuclei of the diplokaryon. Yeast two-hybrid and co-immunoprecipitation analysis verified that NbSeptins can interact with each other. Our findings suggest that NbSeptins can cooperate in the proliferation stage of Nosema bombycis and contribute towards the understanding of the rols of septins in microsporidia development.

Nosema sp. PM-1, a new isolate of microsporidian from infected Papilio machaon Linnaeus, based on ultrastructure and molecular identification.

A new microsporidium, Nosema sp. PM-1, was first isolated from Papilio machaon Linnaeus. The spore shape of the PM-1 isolate was a long oval with an average size of 3.22 μm × 1.96 μm. Ultrastructure observation showed that PM-1 had a typical Nosema common diplokaryotic nuclei structure with 10-13 polar filament coils, spore wall, plasma membrane, and anchoring disk. The complete rRNA gene sequences were obtained by polymerase chain reaction amplification and each rRNA unit was arrayed as follows: 5'-LSU (2497 bp)-ITS (179 bp)-SSU (1232 bp)-IGS (278 bp)-5S (115 bp)-3', which was the same as typical Nosema. The phylogenetic trees of rRNA, DNA-directed RNA polymerase II subunit, and tubulin genes all show that PM-1 was a sister to the clade comprising Nosema bombycis, Nosema spodopterae, and Nosema sp. PX1. The spore morphology, ultrastructure, and complete rRNA structure indicate that this isolate assigned to the ˝true˝ Nosema group, can parasitized in Papilio machaon Linnaeus, which provides a wider host range for Nosema.

Molecular and ultrastructural characterization of Andreanna caspii n. gen., n. sp. (Microsporida: Amblyosporidae), a parasite of Ochlerotatus caspius (Diptera: Culicidae)

A new genus and species of microsporidia, Andreanna caspii n. gen., n. sp. is described from the mosquito, Ochlerotatus caspius (Pallas) based on ultrastructural morphology, developmental characteristics, and comparative sequence analyses of the small subunit (SSU) ribosomal DNA (rDNA). Parasite development is confined to fat body tissue and infected larvae appear swollen with dull white masses within the thorax and abdomen. Meronts have diplokaryotic nuclei and are delineated by a simple plasmalemma contiguous with the host cell cytoplasm. Merogony occurs by synchronous binary division followed by cytokinesis. Diplokaryotic sporonts undergo meiosis and synchronous nuclear division forming sporogonial plasmodia with two, four and eight nuclei enclosed within a persistent sporophorous vesicle. Cytokinesis of sporogonial plasmodia results in the formation of eight uninucleate spores. The episporontal space of early sporonts is filled with a homogeneous accumulation of electron dense granular inclusions and ovoid vesicles of various dimensions, transforming into an interwoven matrix during the initial phase of sporogenesis. Spores are oval, uninucleate and measure 4.8 ± 0.3 × 3.1 ± 0.4 μm (fixed). The spore wall is 260 μm thick with an irregular exospore consisting of two layers (150–170 μm) and a thinner endospore (90–100 μm). The anchoring disk is well developed and is contiguous with a lamellar polaroplast that occupies the anterior third of the spore and possess more narrow lamellae on the posterior end. The polar filament is gradually tapered and arranged in a single row consisting of six coils ranging from 180 to 150 μm in diameter. The posterior vacuole (posterosome) is moderately sized and filled with a matrix of moderate electron density. Phylogenetic analysis of SSU rDNA from A. caspii and 30 other species of microsporidia including 11 genera parasitic in mosquitoes using maximum parsimony, neighbor joining and maximum likelihood methods showed A. caspii to be a sister group to the clade containing all of the Amblyospora species, including Culicospora, Edhazardia and Intrapredatorus, as well as Culicosporella and Hyalinocysta thus providing strong support for establishment of Andreanna as a separate genus.

Morphological and molecular characterization of a new microsporidian (Protozoa: Microsporidia) isolated fromSpodoptera litura(Fabricius) (Lepidoptera: Noctuidae)

A microsporidium was isolated from larvae of Spodoptera litura (Fabricius) collected from Tamil Nadu, India. This microsporidian species is monomorphic, disporous and develops in direct contact with the cytoplasm of the host cell. The nuclear configuration of merogonic and sporogonic stages was diplokaryotic. The merogonic proliferative stage was unusual that normal development with 1, 2 and 4 binucleated forms were common, while large multinucleate meronts containing 8 and 12 small compact horseshoe-like diplokaryotic nuclei were also observed. The fresh spores were typically ovocylindrical in shape, with a mean size of 3.91 x 1.91 microm and the polar filament length was approximately 90 microm. Infection was systemic with mature spores produced in the midgut, nervous tissue, muscles, labial glands, gonads, tracheae, epidermis, Malpighian tubules and, most extensively, fat body tissues. The new isolate was highly pathogenic to S. litura larvae. Host specificity tests performed on 37 non-target hosts of 5 different insect orders revealed that the new isolate is pathogenic only to lepidopteran insects. We sequenced the 16S small subunit rRNA (SSU rRNA) gene of the isolate and compared it with 72 non-redundant microsporidian sequences from the GenBank. Based on the light microscopic studies and phylogenetic analyses, the new isolate is assigned to the genus Nosema. Significant differences in the SSU rRNA sequence were identified when compared with the type species Nosema bombycis and other closely related species viz., Nosema spodopterae. Structural differences were also observed in the 16S SSU rRNA between the new isolate and the two above-mentioned microsporidian pathogens. We conclude that the microsporidian isolate reported here is distinctly different from the other known species and is likely to be a new species.

The early events of Brachiola algerae (Microsporidia) infection: spore germination, sporoplasm structure, and development within host cells

Brachiola algerae (Vavra et Undeen, 1970) Lowman, Takvorian et Cali, 2000, originally isolated from a mosquito, has been maintained in rabbit kidney cells at 29 degrees C in our laboratory. This culture system has made it possible to study detailed aspects of its development, including spore activation, polar tube extrusion, and the transfer of the infective sporoplasm. Employing techniques to ultrastructurally process and observe parasite activity in situ without disturbance of the cultures has provided details of the early developmental activities of B. algerae during timed intervals ranging from 5 min to 48 h. Activated and nonactivated spores could be differentiated by morphological changes including the position and arrangement of the polar filament and its internal structure. The majority of spores extruded polar tubes and associated sporoplasms within 5 min post inoculation (p.i.). The multilayered interlaced network (MIN) was present in extracellular sporoplasms and appeared morphologically similar to those observed in germination buffer. Sporoplasms, observed inside host cells were ovoid, contained diplokaryotic nuclei, vesicles reminiscent of the MIN remnants, and their plasmalemma was already electron-dense with the "blister-like" structures, typical of B. algerae. By 15 min p.i., the first indication of parasite cell commitment to division was the presence of chromatin condensation within the diplokaryotic nuclei, cytoplasmic vesicular remnants of the MIN were still present in some parasites, and early signs of appendage formation were present. At 30 min p.i., cell division was observed, appendages became more apparent, and some MIN remnants were still present. By two hours p.i., the appendages became more elaborate and branching, and often connected parasite cells to each other. In addition to multiplication of the organisms, changes in parasite morphology from small oval cells to larger elongated "more typical" parasite cells were observed from 5 h through 36 h p.i. Multiplication of proliferative organisms continued and sporogony was well underway by 48 h p.i., producing sporonts and sporoblasts, but not spores. The observation of early or new infections in cell cultures 12-48 h p.i., suggests that there may also exist a population of spores that do not immediately discharge, but remain viable for some period of time. In addition, phagocytized spores were observed with extruded polar tubes in both the host cytoplasm and the extracellular space, suggesting another means of sporoplasm survival. Finally, extracellular discharged sporoplasms tightly abutted to the host plasmalemma, appeared to be in the process of being incorporated into the host cytoplasm by phagocytosis and/or endocytosis. These observations support the possibility of additional methods of microsporidian entry into host cells and will be discussed.

Chronic microsporidial stromal keratitis in an immunocompetent, non-contact lens wearer

An 82-year-old healthy man with unilateral chronic stromal keratitis, initially diagnosed to have viral keratitis and refractory to medical therapy, showed numerous oval, microsporidial organisms, measuring 4-5 m in length in the corneal biopsy. Penetrating keratoplasty, followed by treatment with systemic albendazole and topical propamidine isethionate resulted in resolution of the infection. Electron microscopy of the keratoplasty specimen demonstrated sporoblasts with diplokaryotic nuclei and multiple coils of the filament. The light and electron microscopic features were consistent with microsporidial keratitis.

Transfer of Nosema locustae (Microsporidia) to Antonospora locustae n. comb. based on molecular and ultrastructural data.

Nosema locustae is a microsporidian parasite of grasshopper pests that is used as a biological control agent, and is one of the emerging model systems for microsporidia. Due largely to its diplokaryotic nuclei, N. locustae has been classified in the genus Nosema, a large genus with members that infect a wide variety of insects. However, some molecular studies have cast doubt on the validity of certain Nosema species, and on the taxonomic position of N. locustae. To clarify the affinities of this important insect parasite we sequenced part of the rRNA operon of N. locustae and conducted a phylogenetic analysis using the complete small subunit rRNA gene. Nosema locustae is only distantly related to the nominotypic N. bombycis, and is instead closely related to Antonospora scoticae, a recently described parasite of bees. We examined the ultrastructure of mature N. locustae spores, and found the spore wall to differ from true Nosema species in having a multi-layered exospore resembling that of Antonospora (one of the distinguishing features of that genus). Based on both molecular and morphological evidence, therefore, we propose transferring N. locustae to the genus Antonospora, as Antonospora locustae n. comb.

Nosema like organisms found in an immunocompetent human patient: First report in Central América

Stool samples of a immunocompetent hypertense 61-year old woman were stained by the Weber et al. method and studied by electronic microscopy. Mature microsporidia spores were found and according to their diplokaryotic nuclei, as well as the typical polar filament with 12 coils, the organisms was classified as Nosema like genus.

Light and Electron Microscopy Studies on the Development of Parathelohania anophelis (Microspora: Amblyosporidae) in Female Anopheles quadrimaculatus (Diptera: Culicidae)

A light and electron microscopic study of Parathelohania anophelis, a heterosporous microsporidian parasite of the mosquito Anopheles quadrimaculatus, revealed both oval and cylindrical binucleate spores in adult females. Both types of spores may be responsible for transovarial transmission of the parasite. Both spore types originate from identical diplokaryotic meronts in larvae. However, the small oval spore results from plasmodia containing 10 to 40 diplokaryotic nuclei and is produced 24 hr after emergence of the adult mosquito, whereas the large cylindrical spore results from plasmodia containing 6 to 10 nuclei and is formed only after the mosquito takes a blood meal.

Nuclear division and chromosome cycle in microsporidia

Nuclear division and chromosome cycle of microsporidia are reviewed in the light of recent speculation that the group diverged as an early branch in the eukaryotic line of descent. Microsporidia are primitive eukaryotis with simple cytoplasmic organisation, lacking mitochondria, peroxisomes and a classical Golgi apparatus. The ribosomes resemble prokaryotic ribosomes in size and in having sequences complementary to the eukaryotic 5.88 rRNA contained within their large (28S) ribosomal subunit, rather than a separate 5.8S molecule. Nuclei may be isolated or closely appressed as a diplokaryotic pair which divide synchronously. Mitosis is intranuclear: there are no centrioles but spindle termini are electron dense plaques in pores in the nuclear envelope. Some genera have isolated nuclei throughout the life cycle, while others have diplokaryotic nuclei throughout: meiosis is not known in either of these categories of genera. In contrast, certain polymorphic species, which are transmitted horizontally between copepods and mosquitoes and vertically between generations of mosquitoes, alternate between stages with isolated nuclei and stages with diplokaryotic nuclei. Haploid spores in copepods are infective to mosquito larvae, in which gametogenesis and plasmogamy occur to give diplokaryotic (diploid) stages. Stages remain diplokaryotic through transovarial transmission to the next generation, when an unusual form of meiosis is initiated in both nuclei of the diplokaryon (as indicated by synaptonemal complexes), and mingling of chromosomes occurs when the two nuclei fuse during pachytene.

Ultrastructural Study and Description of Ovavesicula popilliae N. G., N. Sp. (Microsporida: Pleistophoridae) from the Japanese Beetle, Popillia japonica (Coleoptera: Scarabaeidae)1

ABSTRACTA new genus and species of microsporidia, Ovavesicula popilliae n. g., n. sp., is described from the Japanese beetle, Popillia japonica, on the basis of studies by light and electron microscopy. Parasite development primarily occurs within the Malpighian tubules of larvae, and spores are formed in a sporophorous vesicle. Meronts have diplokaryotic nuclei, develop in direct contact with the host cell cytoplasm, and divide by binary fission. Sporonts have unpaired nuclei, develop within a thick sporophorous vesicle, and undergo synchronous nuclear divisions producing plasmodia with 2, 4, 8, 16, and 32 nuclei. Cytokinesis of sporogonial plasmodia does not occur until karyokinesis is complete with 32 nuclei. Intact sporophorous vesicles are ovoid, containing numerous secretory products, and are surrounded by a persistent two‐layered wall. The uninucleate spores are regularly formed in groups of 32, and the polar tube in each has six coils.