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.     

Temperature-Dependent Spore Dimorphism in Burenella dimorpha (Microspora: Microsporida)

Burenella dimorpha, a microsporidian parasite of the tropical fire ant, Solenopsis geminata, produces two morphologically distinct types of spores. The binucleate free spores (spores not bound by a pansporoblast membrane) develop normally at temperatures at least as low as 20°C and as high as 32°C. The uninucleate octospores (spores bound in octets by a pansporoblast membrane), however, develop in a restricted range of temperature. Octospores constituted 35.9%± 2.6 of the spores in 25 pupae held at 28°C. Raising the temperature to 30°C reduced octospores to < 1% of the total spore population. Lowering the temperature to 25° or 22°C reduced the octospore population to 8.5%± 6.5 or 0.4 ± 0.5, respectively. Inhibition of octospore development was complete at 20°C. In contrast, the octospores of Vairimorpha necatrix and Vairimorpha plodiae are reported to be abundant at 16°C and 21°C, respectively. The critical event blocked in octospore development may be meiosis, as evidenced by an abundance of binucleate sporonts in the octospore sequence of development, and absence of more advanced sporogonic stages in hosts held at inhibitory temperatures. Free spore size is not affected by temperature although yield may be slightly reduced at elevated temperature.     

Spore Dimorphism in a Microsporidan Isolate*

A microsporidan isolate currently considered to represent a mixed infection of Nosema necatrix Kramer, 1965 and Thelohania diazoma Kramer, 1965 was subjected to cultivation in hosts held at various temperatures. The ratio of the Nosema (monospore) to the Thelohania (octospore) forms at these temperatures was found to vary from 1:1 at 16 C to 1:0 at 32 C. Isolation technics using mechanical, temperature and temporal methods separated monosporous from octosporous forms for inoculation purposes. However, microscopic examination of hosts receiving these inocula revealed the presence of both monospores and octospores. Electrophoretic analysis of monospores and monospore-octospore mixtures indicated equivalent hydrophobic protein spectra. These observations suggest that this isolate has the ability to produce either single spores or spores in groups of eight. This microsporidan was not considered a member of the genus Stempellia since spores in groups of 2 or 4 were not observed. Retention of the name Nosema necatrix Kramer is suggested.     

Vairimorpha invictae N. Sp. (Microspora: Microsporida), a Parasite of the Red Imported Fire Ant,Solenopsis invicta Buren (Hymenoptera: Formicidae)12

ABSTRACT. Vairimorpha invictae n. sp. infects the red imported fire ant, Solenopsis invicta Buren, in Brazil. The parasite is dimorphic, producing two morphologically distinct types of spores, which develop sequentially in the same fat cells or oenocytes in the fat body. The binucleate free spores develop from disporous sporonts; the uninucleate octospores develop from multinucleate sporonts within a sporophorous vesicle. Infected cells are transformed into large sacs which contain both types of spores in mature adult hosts. Mature free spores are often present by the time the larvae pupate, but mature octospores are found only in adult hosts. Masses of spores may be seen through the intact cuticle by low power phase-contrast microscopy; there are no other physical signs and no behavioral signs of infection. Attempts to transmit the infection in the laboratory failed.     

Identification of a microspordium isolated from Megacopta cribraria (Hemiptera: Plataspidae) and characterization of its pathogenicity in silkworms

A new microsporidium isolated from Megacopta cribraria was characterized by both biological characteristics and phylogenetic analysis. Moreover, its pathogenicity to silkworms was also studied. The spores are oval in shape and measured 3.64 ± 0.2 × 2.20 ± 0.2 μm in size. Its ultrastructure is characteristic of the genus Nosema: a diplokaryon, 13–14 polar filament coils and posterior vacuole. Its life cycle includes meronts, sporonts, sporoblasts and mature spores, with a typical diplokaryon in each stage and propagation in a binary fission. A phylogenetic tree based on SSU rRNA and rRNA ITS gene sequence analysis further indicated that the parasite is closely related to Nosema bombycis and should be placed in the genus Nosema and sub-group ‘true’ Nosema. Furthermore, the microsporidium heavily infects lepidopteran silkworm insect and can be transmitted per os (horizontally) and transovarially (vertically). Our findings showed that the microsporidium belongs to the ‘true’ Nosema group within the genus Nosema and heavily infects silkworms. Based on the information obtained during this study, we named this new microsporidium isolated from M. cribraria as Nosema sp. MC.     

Phylogenetic relationships of Heterovesicula cowani, a microsporidian pathogen of Mormon crickets, Anabrus simplex (Orthoptera: Tettigoniidae), based on SSU rDNA-sequence analyses

The microsporidium Heterovesicula cowani, discovered in 1985, was initially identified as Vairimorpha sp. because it produces two types of spores: Nosema-like diplokaryotic spores and Thelohania-like mononuclear meiospores. However, light and electron microscopy studies revealed characters that did not fit any known microsporidian genera, and a new monotypic genus Heterovesicula was erected. The goal of this study was to test the validity of the genus Heterovesicula by molecular characterization of H. cowani and to assess its phylogenetic relationships to other microsporidia from insects. DNA from spores stored at −32 °C since 1992 was isolated and PCR-amplified with V1-1492 primers to obtain a partial small subunit ribosomal RNA gene sequence of 1165 bp, which was submitted to GenBank (Accession No. EU275200). Neighbor joining, maximum parsimony and maximum likelihood analyses performed against 18 microsporidia sequences, placed H. cowani as a sister taxon to the Nosema–Vairimorpha clade. The consensus of these analyses suggests that the Heterovesicula–Nosema–Vairimorpha group forms a dichotomy with the Encephalitozoon spp. branch. Other microsporidia parasitizing Orthoptera fell into two unrelated (or distantly related) lineages of terrestrial microsporidia: the Liebermannia spp. branch forms a dichotomy with Orthosomella operophterae within the Endoreticulatus–Orthosomella–Liebermannia group; and the Paranosema spp. branch clusters together with the Tubulinosema–Systenostrema lineage. The minimum pairwise distance in Kimura-2-Parameter analysis among 18 analyzed sequences was 0.37, which supports well the generic status for Heterovesicula. The obtained phylogenetic trees suggest that H. cowani is related to the Vairimorpha necatrix group, but not to other insect microsporidia producing octospores.     

A new mycosis of larval lobster (Homarus americanus).

Spodoptera exempta larvae were reared on semisynthetic maize diet. Pathogenicity studies were undertaken on first- to fifth-instar larvae fed a high dosage of Nosema necatrix spores. Larvae from the earlier instars were most susceptible to the microsporidan and also developed bacteriosis. A cytoplasmic polyhedrosis virus (CPV) was evident in some infected larvae but not in controls. The development of N. necatrix is redescribed using the light microscope. A disporoblastic life cycle was evident at 25°C and both a disporoblastic and an octosporoblastic life cycle at 20°C. The implications of the occurrence of bacteriosis and CPV and the possible biological significance of the two sporogonic sequences are discussed. The taxonomic position of N.necatrix is reviewed and, after comparison with existing species of the genera Nosema and Parathelohania, it is placed in the new genus Vairimorpha. The implications of polymorphism are discussed in relation to the classification of the Microsporida.     

Genus Pleistophora Gurley, 1893: An Assemblage of at Least Three Genera1

Until recently, pansporoblastic microsporidia that produce a variable and large number of sporoblasts from a sporont have been included in a single genus, namely Pleistophora Gurley, 1893. Ultrastructural studies have been used to determine whether the resemblance of these species is fundamental or superficial. The results indicated that the multisporous pansporoblastic forms belong to at least three genera and, thus, that Pleistophora is a “composite genus.” The term pansporoblast was originally used for stages in myxosporidian development. The term sporophorous vesicle adopted from Gurley is suggested for the spore-containing vesicle in the Microspora. Three species were studied: Pleistophora typicalis, the type-species; Pleistophora culicis, for which a new genus Vavraia has already been proposed; and Pleistophora simulii. P. typicalis and V. culicis have isolated nuclei throughout their development, and the sporophorous vesicle wall enveloping the sporoblasts is derived from amorphous secretions laid down during merogony external to the plasmalemma. Pleistophora and Vavraia are differentiated principally in terms of the structure of the sporophorous vesicle wall and mode of division of the sporogonial plasmodium. The nuclei of young sporonts of P. simulii are in diplokaryon arrangement and undergo meiosis to give haploid nuclei in the sporoblasts. The sporophorous vesicle wall is membranoid and is laid down external to the plasmalemma at the onset of sporogony. A new genus, Polydispyrenia n. g., is suggested for this species, the affinities of which are closer to the dimorphic species of microsporidia than to Pleistophora or Vavraia. The terms “merontogenetic sporophorous vesicle” and “sporontogenetic sporophorous vesicle” are used to distinguish between the two groups.     

The ultrastructure of three microsporidia from winter moth,Operophtera brumata (L.), and the establishment of a new genus Cystosporogenes n.g. for Pleistophora operophterae (Canning, 1960)

Summary The ultrastructure of three species of microsporidia in winter moths, Operophtera brumata (L.), has been used to consolidate taxonomic assessments previously based on light microscopy. The characters formerly used to assign Nosema operophterae Canning, 1960 to a new genus Orthosoma Canning, Wigley & Barker, 1983, namely that the nuclei are isolated and that sporoblasts are separated from ribbon-shaped multinucleate (2, 4, 8 or rarely 12 nuclei) sporonts, were upheld at the ultrastructural level. Development was in contact with the cell cytoplasm but all stages, which must have included meronts, had an electron dense surface coat. Nosema wistmansi Canning, Wigley & Barker, 1983, was found to be ultrastructurally typical of the genus Nosema Naegeli, 1857. An unusual feature of this species was the close association of cysternae of host endoplasmic reticulum with the surface of meronts, an association lost in sporogony. Pleistophora operophterae (Canning, 1960) has been transferred, on ultrastructural criteria, to a new genus Cystosporogenes n.g. Nuclei are isolated; all stages develop in a vesicle bounded by an envelope of enigmatic origin; this envelope persists around the spores as a sporophorous vesicle; division of the sporont within this vesicle is by budding and the number of sporoblasts, and therefore spores, is variable up to about 60.Microsporidia which undergo multisporous sporogony in sporophorous vesicles are now distributed among seven genera. These are: Glugea Thélohan, 1891; Pleistophora Gurley, 1893; Pseudopleistophora Sprague, 1977; Vavraia Weiser, 1977; Baculea Loubès & Akbarieh, 1978; Polydispyrenia Canning & Hazard, 1982 and Cystosporogenes n.g. New genera would appear to be needed for Pleistophora sp. of Sandars & Poinar (1976) and Pleistophora sp. of Percy, Wilson & Burke (1982). ac]19840404     

Pathologie d’une microsporidiose de l’arpenteuse du chou,Trichoplusia ni [Lep.: Noctuidae], parVairimorpha necatrix

Résumé Les larves deTrichoplusia ni (Hübner) infectées parVairimorpha (=Nosema) necatrix (Kramer) présentent des sympt?mes chroniques, semi-chroniques et aigus selon les doses de spores deV. necatrix (5 à 500, 5.10³ à 5.10⁵ et 5.10⁶ par larve, respectivement) ingérées par larve.V. necatrix parasite principalement le corps adipeux et les tissus des muscles quand les larves ont des sympt?mes chroniques, tandis que les larves manifestent les sympt?mes aigus lorsque les microsporidies attaquent principalement l’intestin moyen. L’histopathologie de la maladie est étudiée.

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Summary Infection of larvae ofTrichoplusia ni (Hübner) byVairimorpha, (=Nosema) necatrix was classified as chronic, semi-chronic and acute symptoms depending on the quantity of spores (5 to 500, 5×10³ to 5×10⁵, and 5×10⁶ per larva, respectively) ingested per larva.V. necatrix infects mainly the fat body and some muscle tissue of larvae with chronic symptoms and mainly midgut tissue of larvae with acute symptoms.V. necatrix caused death in 3 to 4.5 days when a larva ingested 5×10⁶ spores. The histopathology of the disease was studied.

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Ce travail a fait l’objet d’une thèse de 3^e cycle, présentée à l’Université de Paris VI en mars 1977, parWei Hsuang Chu.     

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.     

Ultrastructural Study of the Development of Pleistophora schubergi Zwölfer, 1927 (Protozoa,Microsporida) in Larvae of the Spruce Budworm,Choristoneura fumiferana and Its Subsequent Taxonomic Change to the Genus Endoreticulatus

This study demonstrates that Pleistophora schubergi Zwölfer, 1927, a microsporidium originally isolated from the midgut epithelium of Nygmia phaeorrhoea Don (Euproctis chrysorrhoea L.) and Porthetria dispar L., and subsequently reported in several other insects including the spruce budworm, Choristoneura fumiferana (the host used in this investigation), does not belong in the genus Pleistophora Gurley, 1893. Pleistophora schubergi lacks the major features that are characteristic of Pleistophora typicalis, the type species of this genus. A comparison of ultrastructural observations reported for the type species of the genus Pleistophora, P. typicalis, and our observations of P. schubergi revealed significant differences. A thick (0.5 μm) amorphous coat, derived from parasite secretions and deposited external to the parasite plasmalemma, surrounds all developmental stages in P. typicalis. Double membranes, derived from host rough endoplasmic reticulum cisternae encircle the parasite plasmalemma of all developmental stages in P. schubergi. The sporophorous vesicle encases the spores in P. typicalis, and originates from the parasite-secreted coat that is present around meronts. In P. schubergi, the host endoplasmic reticulum cisternae form the envelope that surrounds the meronts. Moreover, the sporophorous vesicle envelope in P. typicalis persists around groups of spores, while in P. schubergi this envelope breaks easily to release the spores in the host cytoplasm. By comparing the characteristics of the microsporidium found in the spruce budworm with those of the recently created polysporous genera that sporulate within a vesicle, we found that P. schubergi does belong in the new genus Endoreticulatus Brooks et al. 1988, and consequently rename it Endoreticulatus schubergi (Zwölfer, 1927) n. comb.     

Microsporidia of the Genus Trachipleistophora—Causative Agents of Human Microsporidiosis: Description of Trachipleistophora anthropophthera N. Sp. (Protozoa: Microsporidia)

Trachipleistophora anthropophthera n. sp., was found at autopsy in the brain of one and in the brain, kidneys, pancreas, thyroid, parathyroid, heart, liver, spleen, lymph nodes, and bone marrow of a second patient with AIDS. The parasite is similar to the recently described T. hominis Hollister, Canning, Weidner, Field. Kench and Marriott, 1996, in having isolated nuclei, meronts with a thick layer of electron dense material on the outer face of their plasmalemma and sporogony during which spores are formed inside a thick-walled sporophorous vesicle. In contrast to T. hominis , this species is dimorphic as it forms two kinds of sporophorous vesicles and spores: Type I-round to oval polysporous sporophorous vesicle. 7-10 μm in size, usually with eight spores (3.7 × 2.0 μm), thick endospores, subterminal anchoring disc and anisofilar polar filaments forming seven thicker and two thinner terminal coils. This type of sporophorous vesicle is associated with 25-30 nm filaments extending into the host cell cytoplasm. Type II—smaller, bisporous sporophorous vesicle (4-5 times 2.2-2.5 μm) with two, nearly round, thin-walled spores, 2.2-2.5 × 1.8-2.0 μm in size, having 4-5 isofilar coils. No outside filamentous elements are associated with the bisporous sporophorous vesicle. Both types of sporophorous vesicles were common in the infected brain tissue and could be found within the same cell. The newly described species, together with T. hominis and previously reported Pleistophora -like parasites from human muscle, likely represent a group of closely related human microsporidia.     

Napamichum cellatum N. Sp. (Microspora, Thelohaniidae), a New Parasite of Midge Larvae of the Genus Endochironomus (Diptera, Chironomidae) in Sweden

ABSTRACT The new microsporidium, Napamichum cellatum, a parasite of the adipose tissue of midge larva of the genus Endochironomus in Sweden, is described based on light microscopic and ultrastructural characteristics. Plurinucleate Plasmodia with nuclei arranged as diplokarya divide, probably by plasmotomy, producing a small number of diplokaryotic merozoites. The number of merogonial cycles is unknown. Each diplokaryotic sporont yields eight monokaryotic sporoblasts in a thin-walled, more or less fusiform sporophorous vesicle. A small number of multisporoblastic sporophorous vesicles were observed, in which a part of the sporoblasts were anomalous. The sporogony probably begins with a meiotic division. The mature spores are slightly pyriform. Fixed and stained spores measure 2.1-2.4 × 3.7-4.5 μm. The five-layered spore wall is of the Napamichum type. The polar filament is anisofilar with seven to eight coils (142-156 and 120 nm wide). The angle of tilt is 55-65°. The polaroplast has an anterior lamellar and a posterior tubular part. The granular, tubular and crystal-like inclusions of the episporontal space disappear more or less completely when the spores mature. The crystal-like inclusions are prominent in haematoxylin staining, but not visible with the Giemsa technique. The microsporidium is compared to other octosporoblastic microsporidia of midge larva and to the species of the genera Chapmanium and Napamichum.     

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.     

A new isolate of Nosema sp. (Microsporidia, Nosematidae) from Phyllobrotica armata Baly (Coleoptera, Chrysomelidae) from China

We studied the spore morphology and molecular systematics of a novel microsporidian isolate from Phyllobrotica armata Baly collected in China. The spores were long-oval and measured 4.7 × 2.6 μm on fresh smears. Ultrastructure of the spores was characteristic for the genus Nosema: 13-14 polar filament coils, posterior vacuole, and a diplokaryon. The complete rRNA gene sequence of the isolate was 4308 bp long. The organization of the rRNA gene was 5′-LSU rRNA-ITS-SSU rRNA-IGS-5S-3′, which corresponds to that of the Nosema species. Phylogenetic analysis based on the rRNA gene sequence indicated that this isolate, designated as Nosema sp. PA, is closely related to Nosemabombycis and is correctly assigned to the “true” Nosema group.     

Mass production and storage of Vairimorpha necatrix (protozoa: Microsporida)

Mass production and storage methods were evaluated for maximization of spores of Vairimorpha necatrix, a promising protozoan for microbial control due to its virulence and prolificity in lepidopterous pests. In vivo spore production was at a maximum when 3rd instar Heliothis zea were exposed to 6.6 spores/mm² of artificial diet surface and reared for 15 days. Approximately 1.67 × 10¹⁰ spores/larva were produced, or ca. 1 × 10¹⁰ spores/larva after partial purification of the spores by homogenization of the larvae in water, filtration, and centrifugation. The spores were inactivated by relatively short exposures to several chemicals which were tested to counteract contamination of the diet surface by fungi in the spore inoculum. Spores of V. necatrix were stored at refrigerated and freezing temperatures for up to 2 years and bioassayed periodically with 2nd instar H. zea. Spores lost little infectivity after 23 months at 6°C if they were stored in a purified water suspension plus antibiotic, but they were noninfective after 18 months at 6°C if stored in host tissue. Storage at ?15°C caused little loss of infectivity whether the spores were stored in water and glycerine, in host tissue, or after lyophilization. The spores withstood lyophilization in host cadavers better than in purified water suspension. Samples of a dry V. necatrix-corn meal formulation, which was prepared for field efficacy tests and stored at ?15° and 6°C, were highly infective after 9 months. Large numbers of V. necatrix spores can thus be produced and later made available for microbial control field trials with little loss of infectivity.     

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.