Ultrastructural aspects of a new species,Vavraia mediterranica (Microsporidia,Pleistophoridae), parasite of the French Mediterranean shrimp,Crangon crangon (Crustacea,Decapoda)

The life cycle stages of a new species of the genus Vavraia (Microsporidia, Pleistophoridae), which parasitizes the shrimp Crangon crangon (Crustacea, Decapoda), were examined by light and electron microscopy. This parasite was monomorphic with polysporous sporogony and developed in the skeletal muscle of the host. The multinucleate sporogonial plasmodium divided by plasmotomy and multiple division into uninucleate sporoblasts. All stages were surrounded by a thick and amorphous dense coat external to the plasmalemma. This structure gradually became a merontogenetic sporophorous vacuole (MSV) where the sporonts developed into sporoblasts. The MSV was filled with episporontal granular secretory products and eventually contained up to 50 uninucleate spores. During spore morphogenesis, these episporontal granular products within the MSV became organized as episporontal tubular-like structures. In transverse sections, these structures showed a mean diameter of 1.0 microm, but disappeared during the final phase of the spore maturation. Mature spores were ellipsoidal to slightly pyriform and measured 2.30 x 1.41 microm. The polar filament was anisofilar and consisted of a single coil with six to seven turns (rarely five). This new species is named Vavraia mediterranica n. sp.     

萨梅诺娃新佩雷斯虫中国新记录

研究报道了中国首例摇蚊微孢子虫, 结合各发育阶段形态特征、生态学特征及分子特征, 鉴定其为萨梅诺娃新佩雷斯虫Neoperezia semenovaiae Issi, et al. 2012, 系我国新记录。萨梅诺娃新佩雷斯虫寄生于羽摇蚊幼虫脂肪体组织, 导致其体表呈白浊状。成熟孢子呈卵圆形, 孢子长(5.7±0.2) μm (5.3—6.3 μm), 宽(3.7±0.1) μm (3.4—4.0 μm)。透射电镜观察显示各发育阶段均为离核, 发育不同步, 与宿主细胞质直接接触。早期发育阶段为高电子密度的多核裂殖体阶段, 经原生质团分裂形成单核或多核产孢体, 进一步发育为单核孢子母细胞。孢子母细胞形状不规则, 周围被内质网环绕, 并逐渐形成微孢子虫的典型结构如极丝、极质体和三层孢壁等。成熟孢子卵圆形, 离核, 细胞核较大, 位于孢子正中央, 被大量核糖体包围。极质体分为两部分, 前半部分为海绵状, 后半部分薄膜状。锚状盘位于孢子前端, 呈蘑菇状。孢壁三层, 外层为高电子密度层, 厚26.5—62.7 nm, 中间层为电子透明层, 厚151.8—236.1 nm, 里层为质膜层。同型极丝, 30—31圈, 分2—3列排列。扩增获得其小核糖体序列为1356 bp, 序列比较发现其与俄罗斯列宁格勒区羽摇蚊的N. semenovaiae相似性为99.1%。系统发育关系分析表明N. semonovaiae与Neoperezia、Bryonosema、Schroedera属种类聚为一独立进化枝, N. semonovaiae种群出现明显的地理分化。     

Ultrastructural characteristics and small subunit ribosomal DNA sequence of Vairimorpha cheracis sp. nov., (Microspora: Burenellidae), a parasite of the Australian yabby, Cherax destructor (Decapoda: Parastacidae)

This is the first record of a species of Vairimorpha infecting a crustacean host. Vairimorpha cheracis sp. nov. was found in a highland population of the Australian freshwater crayfish, Cherax destructor. The majority of spores and earlier developmental stages of V. cheracis sp. nov. were found within striated muscle cells of the thorax, abdomen, and appendages of the crayfish. Only octosporoblastic sporogony within sporophorous vesicles (SPVs) was observed. Diplokaryotic sporonts separated into two uninucleate daughter cells, each of which gave rise to four sporoblasts in a rosette-shaped plasmodium, so that eight uninucleate spores were produced within the persistent ovoid SPV. Ultrastructural features of stages in the octosporoblastic sequence were similar to those described for Vairimorpha necatrix, the type species. Mature spores were pyriform in shape and averaged 3.4x1.9 microm in dimensions. The anterior polaroplast was lamellar in structure, and the posterior polaroplast vesicular. The polar filament was coiled 10-12 times, lateral to the posterior vacuole. The small subunit ribosomal DNA (SSU rDNA) of V. cheracis sp. nov. was sequenced and compared with other microsporidia. V. cheracis sp. nov. showed over 97% sequence identity with Vairimorpha imperfecta and five species of Nosema, and only 86% sequence identity with V. necatrix. The need for a taxonomic revision of the Nosema/Vairimorpha group of species is discussed.     

Life Cycles of Myxogastria Stemonitopsis typhina and Stemonitis fusca on Agar Culture

Myxogastria is a group of protozoa characterized by cellular uninucleate amoeboflagellates (myxamoebae and flagellated swarm cell), acellular multinucleate plasmodia, and stationary spore‐bearing sporocarps. The Stemonitales is a large order in the Myxogastria and contains approximately 230 species, but only 13 species have their completed life cycles observed so far. Here, we described the life cycles of two species in Stemonitales, Stemonitopsis typhina and Stemonitis fusca by culturing in water agar medium and observing the morphogenesis of their spore germination, plasmodium, and sporocarp development. The spore‐to‐spore life cycles of Ste. typhina and S. fusca were completed in approximately 67 and 12 d, respectively. Both species possessed an aphanoplasmodium. However, the spores of Ste. typhina and S. fusca germinated by the V‐shape split and pore methods, respectively. Unlike S. fusca with an evanescent peridium, Ste. typhina produced a shiny persistent peridium which was continuous with the membrane surrounding its stalk. The information will contribute to a better understanding of their taxonomy and phylogeny.     

Transmission and infectivity of spores of Burenella dimorpha (Microsporida: Burenellidae)

Burenella dimorpha infects the tropical fire ant, Solenopsis geminata, producing two morphologically distinct types of spores. A binucleate, nonpansporoblast membrane-bounded (NPMB) spore develops in and destroys the hypodermis, rupturing the cuticle in the pupal stage. A uninucleate, pansporoblast membrane-bounded (PMB) spore develops in the fat body. Adult ants cannibalize ruptured pupae but do not ingest spores. Instead, the spores and particulate foods are diverted to the infrabuccal cavity, formed into an infrabuccal pellet, and fed to fourth-instar larvae only. This larval instar is the only stage in the life cycle of S. geminata that is vulnerable to infection. NPMB spores are infective, but PMB spores do not extrude their polar filaments in the larval gut and are expelled in the meconium upon pupation.     

Ameson hybomitrae sp. n. (Microsporidia, Pereziidae) from gadflies of Karelia

Microsporidia of the genus Ameson were recorded from larvae of horseflies of the genus Hybomitra in Karelia. Earlier these Microsporidia were recorded from crustaceans. The infection extensiveness ranges from 5.1 to 10.5%. The parasites develop in musculature, fat body and salivary glands of the host. The new species has uninucleate, single-located egg- and pear-shaped spores. The ultrafine structure of developmental stages and spores is studied.     

Life cycle,ultrastructure and molecular phylogeny of Hyalinocysta chapmani (Microsporidia: Thelohaniidae), a parasite of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae)

The complete life cycle of the microsporidium Hyalinocysta chapmani is described from the primary mosquito host Culiseta melanura and the intermediate copepod host Orthocyclops modestus. Infections are initiated in larval C. melanura following the oral ingestion of uninucleate spores from infected copepods. Spores germinate within the lumen of the midgut and directly invade fat body tissue where all development occurs. Uninucleated schizonts undergo binary division (schizogony) followed by karyokinesis (nuclear division) to form diplokaryotic meronts. Merogony is by synchronous binary division. The onset of sporogony is characterized by the simultaneous secretion of a sporophorous vesicle and meiotic division of the diplokaryon resulting in the formation of eight ovoid meiospores enclosed within a sporophorous vesicle. Most infected larvae die during the fourth stadium and there is no evidence of a developmental sequence leading to vertical transmission. Hyalinocysta chapmani is horizontally transmitted to O. modestus via oral ingestion of meiospores. Infections become established within ovarian tissue of females and all parasite development is haplophasic. Uninucleate schizonts divide by binary division during an initial schizogonic cycle. Newly formed uninucleate cells produce a thin sporophorous vesicle and undergo repeated nuclear division during sporogony to produce a rosette-shaped, multinucleated sporogonial plasmodium with up to 18 nuclei. This is followed by cytoplasmic cleavage, sporogenesis, and disintegration of the sporophorous vesicle to form membrane-free uninucleate spores. Infected females eventually die and there is no egg development. The small subunit rDNA sequence of H. chapmani isolated from meiospores from C. melanura was identical to the small subunit rDNA sequence obtained from spores from O. modestus, corroborating the laboratory transmission studies and confirming the intermediary role of O. modestus in the life cycle. Phylogenetic analysis was conducted with closely related microsporidia from mosquitoes. Hyalinocysta chapmani did not cluster within described Amblyospora species and can be considered a sister group, warranting separate genus status.     

Morphological and molecular comparison of <Emphasis Type="Italic">Myxobolus procerus</Emphasis> (Kudo, 1934) and <Emphasis Type="Italic">M. intramusculi</Emphasis> n. sp. (Myxozoa) parasitising muscles of the trout-perch <Emphasis Type="Italic">Percopsis omiscomaycus</Emphasis>

Inter- and intracellular cysts from a presumed single species, Myxobolus procerus (Kudo, 1934), parasitising the trout-perch Percopsis omiscomaycus were compared with respect to their spore morphology and small subunit 18S ribosomal DNA. The two cyst types have similar pyriform spores and comparable ranges in spore length and width, size of the polar capsules and number of filament coils. However, fixed spores of the intracellular cysts are significantly shorter (p < 0.05) in mean length than those from intercellular cysts, giving them a broader appearance. Comparing 991 base pairs of 18S rDNA revealed a 20 bp difference between the two cyst types (97.9% similarity), 10 due to base substitutions and 10 due to the presence or absence of a base. The described morphological differences and consistent 2.1% difference in the 18S rDNA reveal that the intracellular form is a separate species, which is described herein as Myxobolus intramusculi n. sp. M. intramusculi n. sp. and M. procerus may be sister species and it is suggested that the two species arose through sympatric speciation involving a switch in site specificity within a single host species.     

Meiosis in Microsporidia: effects on biological cycles]

Synaptinemal complexes have been demonstrated in 7 microsporidian species belonging to 6 different genera (Gurleya, Thelohania, Pleistophora, Tuzetia, Baculea, Glugea). Thus, it can be presumed that a meiosis and consequently a karyogamy occur during their life cycle. Meisis occurs at the beginning of sporogony; therefore, karyogamy, must occur between spore and merogany, i.e. during the poorly known part of the life cycle. In the microsporidian species studied, with uninucleate spores and diplokaryotic merogony (Thelohania for instance), the 2 joined nuclei, each of them containing meiotic chromosomes, not only fail to fuse, but actually separate at the beginning of sporogony; afterwards, each of them undergoes meiosis. Their separation is accompanied by the appearance of an organelle whose structure and function are poorly understood. However, its structure resembles that of the kinetic center. The Nosema species studied do not have synaptinemal complexes; thus, their life cycle is difficult to understand: either karyogamy and meiosis occur during the unobserved part of the life-cycle, or sexual phanomena are absent altogether. In the latter case, the Nosema-type life cycle might be limited to vegetative multiplication which could be explained by the dimorphism theory of Microsporidia. It is shown also in the present study that the life cycle of Microsporidia does not involve haploid organisms which it might be thought to contain by comparing it with the cycles of sporozoa.     

Myosporidium merluccius n. g., n. sp. infecting muscle of commercial hake (Merluccius sp.) from fisheries near Namibia

A new species of Microsporidia classified to a new genus was observed in the trunk muscle of commercial hake (Merluccius capensis/paradoxus complex) from Namibian fisheries. Macroscopic examination revealed thin and dark filaments inserted among muscle fibers. Inside the filaments were many sporophorous vesicles with about 30-50 spores per vesicle. The shape of the spore was pyriform and the extruded polar filament was of moderate length (up to 4.29 microm, n=12). This new species of Microsporidia is described using macrophotography, microphotography, staining, and transmission electron microscopy (TEM), as well as molecular methods. Its 16S rRNA was found to be similar to that of Microsporidium prosopium Kent et al., 1999, while both sequences were quite different from 16S rRNA sequences known for other Microsporidia. Nevertheless, this new species is separated morphologically from M. prosopium by the presence of 11-12 anisofilar coils and the formation of the xenoma at the site of infection. Type species.     

Pankovaia semitubulata gen. et sp. n. (Microsporidia: Tuzetiidae) from nymphs of mayflies Cloeon dipterum L. (Insecta: Ephemeroptera) in Western Siberia

The ultrastructure of a new microsporidian, Pankovaia semitubulata gen. et sp. n. (Microsporidia: Tuzetiidae), from the fat body of Cloeon dipterum (L.) (Ephemeroptera: Baetidae) is described. The species is monokaryotic throughout the life cycle, developing in direct contact with the host cell cytoplasm. Sporogonial plasmodium divides into 2-8 sporoblasts. Each sporoblast, then spore, is enclosed in an individual sporophorous vesicle. Fixed and stained spores of the type species P. semitubulata are 3.4 x 1.9microm in size. The polaroplast is bipartite (lamellar and vesicular). The polar filament is isofilar, possessing 6 coils in one row. The following features distinguish the genus Pankovaia from other monokaryotic genera of Tuzetiidae: (a) exospore is composed of multiple irregularly laid tubules with a lengthwise opening, referred to as "semitubules"; (b) episporontal space of sporophorous vesicle (SPV) is devoid of secretory formations; (c) SPV envelope is represented by a thin fragile membrane.     

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.     

Redescription of Microsporidium takedai (Awakura, 1974) as Kabatana takedai (Awakura, 1974) comb. n

Ultrastructural study of the microsporidian Microsporidium takedai from the muscles of masu salmon Oncorhynchus masou proved that this species can be assigned to the genus Kabatana Lom, Dyková and Tonguthai, 2000. The parasites develop within disintegrated sarcoplasm without any delimiting boundary or cyst. Cylindrical multinucleate meronts proliferate by serial constrictions into uninucleate stages which repeat the process. Eventually, the uninucleate stages transform into uninucleate sporonts, which divide once to produce sporoblasts, thus functioning as sporoblast mother cells. Spores, with a subterminally located anchoring disc and 3 to 4 turns of the polar tube coil, average 3.3 by 1.9 microm in size. The exospore is divided into small fields; the endospore frequently makes small invaginations into the spore inside. Phylogenetic analysis using SSU rDNA sequence consistently placed Kabatana takedai in a group consisting of Microgemma sp., Spraguea lophii and Glugea americanus. The K. takedai could easily be separated from the other species in the same group by 2 inserts in the SSU rDNA sequence.     

Phylogenetic affiliation of the desert truffles <Emphasis Type="Italic">Picoa juniperi</Emphasis> and <Emphasis Type="Italic">Picoa lefebvrei</Emphasis>

The molecular phylogeny and comparative morphological studies reported here provide evidence for the recognition of the genus Picoa, an hypogeous desert truffle, in the family Pyronemataceae (Ascomycota, Pezizales). Picoa juniperi and Picoa lefebvrei were reassigned to the genus Picoa based on large subunit (LSU) sequence (28S) rDNA and internal transcribed spacer (ITS) rDNA (including the partial 18S, ITS1, ITS2, 5.8S gene, and partial 28S of the nuclear rDNA) data. Morphological studies of spores, asci, perida, and gleba revealed high similarities between P. lefebvrei and P. juniperi, thereby confirming the membership of both species in the genus Picoa. These two species were primarily distinguishable based on ascospore ornamentation.     

Acaulospora alpina, a new arbuscular mycorrhizal fungal species characteristic for high mountainous and alpine regions of the Swiss Alps

Acaulospora alpina sp. nov. forms small (65-85 microm diam), dark yellow to orange-brown spores laterally on the neck of hyaline to subhyaline sporiferous saccules. The spores have a three-layered outer spore wall, a bi-layered middle wall and a three-layered inner wall. The surface of the second layer of the outer spore wall is ornamented, having regular, circular pits (1.5-2 microm diam) that are as deep as wide and truncated conical. A "beaded" wall layer as found in most other Acaulospora spp. is lacking. The spore morphology of A. alpina resembles that of A. paulinae but can be differentiated easily by the unique ornamentation with the characteristic pits and by the spore color. A key is presented summarizing the morphological differences among Acaulospora species with an ornamented outer spore wall. Partial DNA sequences of the ITS1, 5.8S subunit and ITS2 regions of ribosomal DNA show that A. alpina and A. paulinae are not closely related. Acaulospora lacunosa, which has similar color but has generally bigger spores, also has distinct rDNA sequences. Acaulospora alpina is a characteristic member of the arbuscular mycorrhizal fungal communities in soils with pH 3.5-6.5 in grasslands of the Swiss Alps at altitudes between 1800 and 2700 m above sea level. It is less frequent at 1300-1800 m above sea level, and it so far has not been found in the Alps below 1300 m or in the lowlands of Switzerland.     

Molecular phylogeny and evolution of mosquito parasitic Microsporidia (Microsporidia: Amblyosporidae)

Amblyospora species and other aquatic Microsporidia were isolated from mosquitoes, black flies, and copepods and the small subunit ribosomal RNA gene was sequenced. Comparative phylogenetic analysis showed a correspondence between the mosquito host genera and their Amblyspora parasite species. There is a clade of Amblyospora species that infect the Culex host group and a clade of Amblyospora that infect the Aedes/Ochlerotatus group of mosquitoes. Parathelohania species, which infect Anopheles mosquitoes, may be the sister group to the Amblyospora in the same way that the Anopheles mosquitoes are thought to be the sister group to the Culex and Aedes mosquitoes. In addition, by sequence analysis of small subunit rDNA from spores, we identified the alternate copepod host for four species of Amblyospora. Amblyospora species are specific for their primary (mosquito) host and each of these mosquito species serves as host for only one Amblyospora species. On the other hand, a single species of copepod can serve as an intermediate host to several Amblyospora species and some Amblyospora species may be found in more than one copepod host. Intrapredatorus barri, a species within a monotypic genus with Amblyospora-like characteristics, falls well within the Amblyospora clade. The genera Edhazardia and Culicospora, which do not have functional meiospores and do not require an intermediate host, but which do have a lanceolate spore type which is ultrastructurally very similar to the Amblyospora spore type found in the copepod, cluster among the Amblyospora species. In the future, the genus Amblyospora may be redefined to include species without obligate intermediate hosts. Hazardia, Berwaldia, Larssonia, Trichotuzetia, and Gurleya are members of a sister group to the Amblyospora clades infecting mosquitoes, and may be representatives of a large group of aquatic parasites.