The Light and Electron Microscopic Cytology of Janacekia adipophila N. Sp. (Microspora, Tuzetiidae), a Microsporidian Parasite of Ptychoptera paludosa (Diptera, Ptychopteridae)

ABSTRACT. The microsporidium Janacekia adipophila n. sp., a parasite of Ptychoptera paludosa larvae in Sweden, is described based on light microscopic and ultrastructural characteristics. Merogonial stages and sporonts are diplokaryotic. Merozoites are formed by rosette-like division. Sporonts develop into sporogonial plasmodia with isolated nuclei. These plasmodia give rise to 8–16 sporoblasts by rosette-like budding. A sporophorous vesicle is initiated by the sporogonial plasmodium. Sporoblasts and spores are enclosed in individual sporophorous vesicles. Granular inclusions of the vesicles, visible using light microscopy, discriminate sporogonial stages from stages of the merogony. The monokaryotic, fresh spores are oval with blunt ends, measuring 4.2-6.3 × 9.1-11.2 μm. Macrospores are formed in small numbers. The spore wall has three subdivisions and the exospore is electron-dense. The polaroplast has two parts: closely arranged lamellae anteriorly, wider sac-like compartments posteriorly. The isofilar polar filament, 191–264 nm wide, has 12-13 coils, which are arranged in one layer in the posterior half of the spore. The electron-dense inclusions of the sporophorous vesicle are modified during sporogony, and vesicles with mature spores are traversed by 21–27 nm wide tubules, which connect the exospore with the envelope of the vesicle. The walls of the tubules, the envelope of the vesicles, and the surface layer of the exospore are all identical double-layered structures. The microsporidium is compared to microsporidia of Ptychopteridae and Tipulidae and to related microsporidia of the family Tuzetiidae.     

Description of Hyalinocysta expilatoria n. sp., a microsporidian parasite of the blackfly Odagmia ornata

Hyalinocysta expilatoria n. sp. is described from a larva of Odagmia ornata collected in Sweden. Infection was restricted to the adipose tissue which was transformed into a syncytium. The earliest stage observed was diplokaryotic merozoites, which mature directly into diplokaryotic sporonts. Each sporont produces a sporophorous vesicle (pansporoblast), which persists, also enclosing mature spores. Usually nuclear divisions result in a plasmodium with 8 nuclei, which fragments into 8 sporoblasts, each of which develops into a spore without further division. Occasionally an aberrant number of spores (2, 4, 6) is formed. The spores are pyriform with a flattened area at the posterior pole. Spores in sporophorous vesicles with 8 spores are 4.0–6.0 μm long, in vesicles with 4 spores 4.0–5.0 μm, and in vesicles with 2 spores 7.0–8.0 μm. In some vesicles the spores develop asynchronously, and 2, 4, or 6 mature spores are found together with 6, 4, or 2 immature. There was also a small number of vesicles with supernumerary spores, less than 8 normally developed. The 325–350 nm thick spore wall is composed of three layers. The polar filament is anisofilar with 7 coils in a single layer. The anterior 5–6 coils are wide, the posterior 2-1 thin. The angle of tilt of the anterior filament coil is approximately 50°. The spore has a single nucleus. The sporophorous vesicle is delimited by a thin membrane, also visible in haematoxylin stained preparations. Vesicles with mature spores are void of metabolic inclusions.     

Occurrence of a New Microsporidan: Enterocytozoon bieneusi n. g., n. sp., in the Enterocytes of a Human Patient with AIDS1

A new microsporidium is reported infesting the enterocytes of a Haitian patient with AIDS. The stages observed were diplokaryotic cells, sporogonial plasmodia, unikaryotic sporoblasts, and spores. Neither a sporophorous vesicle (pansporoblastic membrane) nor parasitophorous vacuole were differentiated around the developmental stages, which were in direct contact with the host cell cytoplasm. The polar tube (5-6 coils) was differentiated before fission of the sporogonial plasmodium. The mature spores measured 1.5 m?m × 0.5 m?. The spore wall was very thin as the endospore was absent or poorly differentiated. The organism is named Enterocytozoon bieneusi n. g., n. sp. and is assigned to the suborder Apansporoblastina.     

On the Cytology of Toxoglugea chironomi (Debaisieux, 1931) Jírovec 1936 (Microspora, Thelohaniidae)

ABSTRACT. The light microscopic and ultrastructural characteristics of a microsporidium provisionally identified as Toxoglugea chironomi (Debaiseux, 1931) Jírovec, 1936, is described. It was isolated from oenocytes and adipose tissue of a midge larva of the genus Dicrotendipes . Merozoites are diplokaryotic. The sporogony produces, by fragmentation, eight monokaryotic spores in a sporophorous vesicle. Mature spores are horse-shoe shaped. The total length is about 5.8 μm, the width 0.8-0.9 μm, the external height of the curve 2.3-3.5 μm, and the external width of the curve 3.5-5.2 μm. The polaroplast has lamellar compartments of two types: narrow and closely packed anteriorly, and wider and more loosely arranged posteriorly. The isofilar polar filament is arranged in 8–10 coils in the posterior fourth of the spore. The external nuclear membrane is sometimes continuous with the endoplasmic reticulum. Lamellar and tubular material of exospore construction are present in the episporontal space from the beginning of sporogony. Teratological and normal spores sometimes occur together in the sporophorous vesicle. The identification of the species is discussed and the ultrastructure is compared to Toxoglugea variabilis , the only further species of the genus with known ultrastructural cytology.     

On the Cytology and Taxonomic Position of Nudispora biformis N. G., N. Sp. (Microspora, Thelohaniidae), a Microsporidian Parasite of the Dragon Fly Coenagrion hastulatum in Sweden

The microsporidium Nudispora biformis n. g., n. sp., a parasite of a larva of the damsel fly Coenagrion hastulatum in Sweden, is described based on light microscopic and ultrastructural characteristics. Merogonial stages and sporonts are diplokaryotic. Sporogony comprises meiotic and mitotic divisions, and finally eight monokaryotic sporoblasts are released from a lobed plasmodium. Sporophorous vesicles are not formed. The monokaryotic spores are oval, measuring 1.4–1.8 × 2.8–3.4 μm in living condition. The thick spore wall has a layered exospore, with a median double-layer. The polaroplast has two lamellar parts, with the closest packed lamellae anteriorly. The isofilar polar filament is arranged in 6 (to 7) coils in the posterior half of the spore. Laminar and tubular extracellular material of exospore construction is present in the proximity of sporogonial stages. In addition to normal spores teratological spores are produced. The microsporidium is compared to the microsporidia of the Odonata; its possible relations to the genus Pseudothelohania and to the Thelohania-like microsporidia are discussed. The new genus is provisionally included in the family Thelohaniidae.     

Occurrence of a new microsporidan: Enterocytozoon bieneusi n.g., n. sp., in the enterocytes of a human patient with AIDS

A new microsporidium is reported infesting the enterocytes of a Haitian patients with AIDS. The stages observed were diplokaryotic cells, sporogonial plasmodia, unikaryotic sporoblasts, and spores. Neither a sporophorous vesicle (pansporoblastic membrane) nor parasitophorous vacuole were differentiated around the developmental stages, which were in direct contact with the host cell cytoplasm. The polar tube (5-6 coils) was differentiated before fission of the sporogonial plasmodium. The mature spores measured 1.5 micron X 0.5 micron. The spore wall was very thin as the endospore was absent or poorly differentiated. The organism is named Enterocytozoon bieneusi n. g., n. sp. and is assigned to the suborder Apansporoblastina.     

Establishment of Endoreticulatus N. G. for Pleistophora fidelis (Hostounský & Weiser, 1975) (Microsporida: Pleistophoridae) Based on the Ultrastructure of a Microsporidium in the Colorado Potato Beetle,Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)1,2

A new genus, Endoreticulatus n. g., is described for the microsporidium Pleistophora fidelis (Hostounský & Weiser, 1975) based on light and electron microscopic studies of a microsporidium in the Colorado potato beetle, Leptinotarsa decemlineata (Say). This latter microsporidium is considered to be conspecific with P. fidelis because both isolates have been shown to be infectious for L. decemlineata where infection is limited to the epithelial cells of the midgut; both are haplokaryotic and develop as groups of sporoblasts and spores in subpersistent vacuoles in the host cell. In addition, meronts, sporonts, and spores of each isolate often occur simultaneously in a common cell, and by light microscopy they both appear similar. Ultrastructural studies of the isolate from L. decemlineata revealed that all developmental stages occur in parasitophorous vacuoles derived from cisternae of rough endoplasmic reticulum of the host cell cytoplasm. Based on the unique nature of the parasitophorous vacuole, a new genus, Endoreticulatus, is proposed for P. fidelis. The genus is compared with the other genera whose species undergo multisporous sporogony in sporophorous vesicles. In addition, the nature of the parasitophorous vacuole of Endoreticulatus fidelis (Hostounský & Weiser, 1975) n. comb. is compared with the parasitophorous vacuole known to encase various developmental stages of several other microsporidian species.     

A New Species of Perezia (Microsporida: Pereziidae) from the Argentine Grasshopper Dichroplus elongatus (Orthoptera: Acrididae)1

The new microsporidium (Microsporida: Pereziidae), Perezia dichroplusae n. sp., infects the epithelial cells of the Malpighian tubules of the Argentine grasshopper Dichroplus elongatus. Characteristics of the pathogen include the following: development in direct contact with the host cell cytoplasm; bi-, tetra-, and sometimes multinucleate diplokaryotic meronts, rounded or elongate in shape; unikaryotic sporonts and sporogonial plasmodia, elongate in shape; sporoblasts and spores uninucleated; spores highly variable in size (1.6–6.7 by 1.0–2.7 μm, x?= 3.5 ± 0.09 by 1.5 ± 0.02, n = 100) with eight or fewer polar tube coils and showing a posterior electron-dense inclusion body.     

An Ultrastructural Study of Vairimorpha necatrix (Microspora, Microsporida) with Particular Reference to Episporontal Inclusions During Octosporogony

ABSTRACT. The life cycle of Vairimorpha necatrix was studied by electron microscopy. Disporous development has two distinct stages: 1) diplokaryotic meronts which are actively mitotic, and 2) diplokaryotic sporonts which are distinguished by reduced ribosome density and a thickened plasmalemma. After final division of the sporont, sporoblasts form spores which are ovocylindrical and measure 4.4 ± 0.08 × 2.3 ± 0.05 μ m (mean ± SE). Octosporous development results in eight haploid spores being formed in a sporophorous vesicle. The uninucleate octospores were smaller than the binucleate dispores and the exospore was thicker but less crenulate in outline. Early in octosporogony, tubules are produced from the sporont plasmalemma and electron-dense material accumulates in the episporontal space. The latter may be amorphous, vesiculated, or vacuolated in appearance and in later stages may take a stacked, lamellar form. At sporoblast formation, exospore material coats the plasmalemma and attached tubules; all inclusions in the episporontal space gradually disappear as spores are formed. These secretory products may have application to taxonomic distinction at the species level.     

Morphology and taxonomy of the microsporidium Liebermannia covasacrae n. sp. from the grasshopper Covasacris pallidinota (Orthoptera, Acrididae)

During a survey for grasshopper pathogens in Argentina in 2005-2006, individual Covasacris pallidinota from halophylous grasslands in Laprida, Buenos Aires province were found to be infected with a microsporidium. Infection was restricted to the salivary gland epithelial cells. The microsporidium produced ovocylindrical spores averaging 2.6 ± 0.28 × 1.4 ± 0.12 μm (range 2.2-3.4 × 1.1-1.7 μm), which resembled in size and shape the spores of Liebermannia patagonica and L. dichroplusae, two recently described species that also parasitize Argentine grasshoppers. The life cycle of the microsporidium included the formation of polynucleate, diplokaryotic, moniliform, merogonial plasmodia wrapped in flattened cisterns of the host endoplasmic reticulum (ER). Plasmodia divided to produce diplokaryotic cells. The latter underwent elongation, dissociation of diplokarya counterparts, vacuolization, dismantling of the host ER envelope, and deposition of electron-dense material outside the plasma membrane. The resultant binucleate sporogonial plasmodia divided into two uninucleate sporoblasts, which eventually transformed into spores. Uninucleate spores contained a lamellar polaroplast, embraced by an elongated polar sac, anchoring disc, 3-5 polar filament coils, and a cluster of anastomizing tubules (sporoblast trans-Golgi, posterosome) at the posterior end. Sequence similarity of the SSU rDNA of the newly discovered microsporidium (Genbank accession no. EU709818) to L. patagonica and L. dichroplusae was 99% and 97%, respectively, suggesting that the three species belong to one genus. All three species fell into one clade in SSU rDNA-based phylogenetic trees produced by neighbor joining, maximum parsimony, and maximum likelihood analyses with 100% statistical support. We assign the name Liebermannia covasacrae to this microsporidium. It can be easily differentiated from both congeners by host species, tissue tropism, type of sporogony, and several features of morphology. Comparison of the three Liebermannia spp. demonstrates that the nuclear phase (dikaryotic versus monokaryotic spores) and type of sporogony (polysporous versus disporous) may vary in closely related species.     

Heterovesicula cowani N. G., N. Sp. (Heterovesiculidae N. Fam.), a Microsporidian Parasite of Mormon Crickets, Anabrus simplex Haldeman, 1852 (Orthoptera: Tettigoniidae)

ABSTRACT. Heterovesicula cowani , n. g., n. sp., is a dimorphic microsporidium described from the adipose tissue of the Mormon cricket, Anabrus simplex Haldeman. Proliferation of the microsporidium is by karyokinesis of uninucleate and binucleate cells to form binucleate and tetranucleate cells, respectively. These cells will undergo binary fission (merogony). Ultimately, the meronts undergo karyokinesis without subsequent cytokinesis producing spherical multinucleate plasmodia that are transitional to 2 types of sporogony. Transitional to disporoblastic sporogony, a fragile interfacial envelope delaminates from the plasmodium with morphogenesis to a monfiliform plasmodium consisting of fusiform binucleate diplokaryotic sporonts. These undergo karyokinesis to form tetranucleate diplokaryotic sporonts that undergo cytokinesis during disintegration of the plasmodium into isolated binucleate sporonts. Transitional to octosporoblastic sporogony, multinucleate plasmodia disintegrate into short monofiliform plasmodia of diplokaryotic sporonts which then segregate while undergoing gradual nuclear dissociation (haplosis by nuclear dissociation). These undergo two sequences of karyokinesis and subsequent multiple fission to form eight uninucleate (haploid) sporoblasts in a fusiform arrangement within a persistent envelope. Binucleate spores are ovocylindrical, about 5.4 × 1.7 μm (fresh), with an isofilar polar filament singly coiled about 11 turns. Uninucleate spores are ovoid to slightly pyriform, 4.0 × 1.7 μm (fresh), with an isofilar filament singly coiled about 9 turns. A new family, Heterovesculidae, is proposed for the new genus.     

Life Cycle of Culicosporella lunata (Hazard & Savage, 1970) Weiser, 1977 (Microspora) as Revealed in the Light Microscope with a Redescription of the Genus and Species1

Light microscopy studies of Culicosporella lunata (Hazard & Savage), a parasite of the mosquito Culex pilosus (Dyar & Knab), revealed two sporogonial sequences. One sequence begins with diplokaryotic meronts that undergo repeated nuclear divisions to produce sporogonial plasmodia with nuclei in diplokaryotic arrangement. These plasmodia form rosette-like clusters of sporoblasts during incomplete cytokinesis and, eventually, binucleate spores. These spores initiate infections in healthy larvae when they ingest spores. The second sequence begins with diplokaryotic meronts that undergo karyogamy and meiosis to form Thelohania-like sporonts and haploid spores. Anomalies are often observed in these sporonts which result in aberrant spores, usually fewer than eight, in an accessory (pansporoblastic) membrane. Normal haploid spores are morphologically similar to those of species of Amblyospora. The genus and the type species are redefined based on new information presented here and it and the type species are placed in the family Amblyosporidae.     

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

A 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.     

Microsporidium novacastriensis n. sp., a Microsporidian Parasite of the Grey Field Slug,Deroceras reticulatum1,2

A new species of microsporidium (phylum Microspora), Microsporidium novacastriensis n. sp., from the grey field slug, Deroceras reticulatum, is described on the basis of light and electron microscope studies. Meronts are spherical at first, then become irregular as nuclear number increases. Sporonts are tubular or ribbon-like and divide unevenly to produce sporoblasts and then spores of varying lengths. Sporogonial stages are enclosed in a vesicle by a subpersistent membrane of uncertain origin. Fresh spores measure 3.5 by 2.08 μm and are produced in clusters of 12 to 120. The parasite infects only the intestinal epithelium of the slug. The new species is compared to microsporidia of other gastropod molluscs and to other microsporidia of similar developmental pattern and morphology.     

A new microsporidian species, Vairimorpha ocinarae n. sp., isolated from Ocinara lida Moore (Lepidoptera: Bombycidae) in Taiwan

A new microsporidium was isolated from Ocinara lida Moore (Lepidoptera: Bombycidae), a pest of Ficus microcarpa L. f. in Taiwan. The microsporidium produces systemic infections in O. lida larvae; the midgut epithelium, Malpighian tubules, and midgut muscle tissues were the target tissues for this isolate, and atrophied fat body tissues were found in heavily infected larvae. Two types of spores were observed, diplokaroytic spores with 11-13 coils of polar tube, and monokaryotic spores with 12 coils of the polar tube that developed within a sporophorous vesicle to form octospores. Electron-dense granules were abundant in the episporontal space of the sporophorous vesicles, and were similar to those of Vairimorpha invictae isolated from Solenopsis invicta, but different from granules or inclusions of other Vairimorpha species. Based on the phylogenetic analysis of the small subunit ribosomal DNA sequence, this isolate is unique within the Vairimorpha complex. Morphological and genetic characters showed this isolate to be a new species. It is placed in the genus Vairimorpha and is described as Vairimorpha ocinarae n. sp.     

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.     

Norlevinea n. g., a New Genus for Glugea daphniae (Protozoa: Microspore), a Parasite of Daphnia longispina (Crustacea: Phyllopoda)1

ABSTRACT. Norlevinea n. g. is established for microsporidia in which a uninucleate meront changes into a sporont by secreting a thin, membranous, sporontogcnetic and fragile sporophorous vesicle (pansporoblast membrane) in which four uninucleate sporoblasts are formed. In contrast to the genus Gurleya, the sporoblasts and later the spores are permanently joined into doublets, being laterally cemented by an electron-dense substance structurally identical to and continuous with the exospore layer. The polar filament is of the anisofilar type. The type species is Norlevinea daphniae (Weiser, 1947) n. comb., a parasite of the ovaries of Daphnia longispina occurring in several carp ponds in Czechoslovakia.     

Development of Edhazardia aedis (Kudo, 1930) n. g., n. comb. (Microsporida: Amblyosporidae) in the mosquito Aedes aegypti (L.) (Diptera: Culicidae)

A microsporidium of the mosquito Aedes aegypti (L.), identified as Nosema aedis Kudo, 1930, was found to be a heterosporous species with 3 sporulation sequences. Usually, 1 sequence developed in a parental generation host individual that was infected per os as a larva and the other 2 developed concurrently in a filial host larva that was infected transovarially. Under some conditions there were deviations from the parental host-filial host alternation. The 1st sporulation sequence was diplokaryotic (diploid in a particular sense) throughout; the other 2 arose from diplokaryotic meronts, developed concurrently and ended with haploid spores. Haplosis in 1 case was by means of dissociation of the diplokaryon. In the other case it was by meiosis. Conflicting reports about whether the members of the diplokaryon in the latter sequence separate and undergo meiosis individually or coalesce and undergo meiosis as 1 nucleus were resolved in favor of the latter idea. A new genus in family Amblyosporidae was created to contain this species, which then became Edhazardia aedis (Kudo, 1930) n. g., n. comb.