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The activities of Tetrahymena corlissi, Tetrahymena thermophila, and Tetrahymena canadensis were studied in coculture with cell lines of insects, fish, amphibians, and mammals. These ciliates remained viable regardless of the animal cell line partner. All three species could engulf animal cells in suspension. However, if the animal cells were monolayer cultures, the monolayers were obliterated by T. corlissi and T. thermophila. Both fibroblast and epithelial monolayers were destroyed but the destruction of human cell monolayers was done more effectively by T. thermophila. By contrast, T. canadensis was unable to destroy any monolayer. At 4 °C T. thermophila and T. corlissi did not carryout phagocytosis and did not destroy monolayers, whereas T. canadensis was able to carryout phagocytosis but still could not destroy monolayers. Therefore, monolayer destruction appeared to require phagocytosis, but by itself this was insufficient. In addition, the ciliates expressed a unique swimming behavior. Tetrahymena corlissi and T. thermophila swam vigorously and repeatedly into the monolayer, which seemed to loosen or dislodge cells, whereas T. canadensis swam above the monolayer. Therefore, differences in swimming behavior might explain why T. corlissi has been reported to be a pathogen but T. canadensis has not., (© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.)

Several new species of trypanosomatids (Euglenozoa, Kinetoplastea, Trypanosomatidae), isolated from the intestines of Neotropical insects (Heteroptera), were genotyped on the basis of spliced leader RNA, and also defined phylogenetically using gene sequences of small subunit ribosomal RNA and glycosomal glyceraldehyde phosphate dehydrogenase. The taxonomic descriptions also included characterization using morphometry and electron microscopy. Our phylogenetic analyses placed the new species within the clade, previously designated "SE" for "Slowly Evolving" sequences of ribosomal RNA genes, a clade that also includes numerous monoxenous parasites of insects from the genera Crithidia, Leptomonas, and Wallaceina, as well as the dixenous genus Leishmania. Based on the high phylogenetic support for this clade, which is consistently recovered in all recent phylogenetic reconstructions, a proposal is put forward to recognize this natural taxon as a new subfamily, Leishmaniinae, within the family Trypanosomatidae., (© 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.)

Brachiola (Nosema) algerae is a microsporidian species generally believed to be an intracellular parasite of insects, especially mosquitoes. However, both mosquito and human isolates have been shown to infect mammalian cells. The present study was undertaken to determine if spores of two insect and two human isolates of B. algerae cultured at 30 degrees C and 37 degrees C differed in their ability to germinate and infect cultured green monkey kidney cells at these two temperatures. Spores from all four isolates exhibited an optimum pH of 9.5 for germination. Mercury (Hg2+) inhibited germination of all isolates equally. Germination of spores from all four isolates was significantly greater when the parasite was cultured at 30 degrees C than when cultured at 37 degrees C. However, spores from the insect isolates cultivated at 30 degrees C or 37 degrees C infected significantly fewer mammalian cells at 37 degrees C than did spores from the human isolates under the same conditions. Thus, there is no correlation between the effects of temperature on the germination and the infectivity of an isolate. In addition, while exposure of B. algerae to 37 degrees C has been reported to cause spore dysmorphism, we failed to observe any consistent ultrastructural changes that explained the greater infectivity of the human isolates at 37 degrees C.

We have determined the sequences of 5S rRNA and spliced leader (SL) RNA genes, and adjacent intergenic regions for representatives of all known trypanosomatid genera parasitizing insects. The genetic loci have been analyzed separately as well as by a combined approach. Several isolates, assigned by morphology to different genera (Leptomonas spp., Blastocrithidia spp.), seem to belong to a single species with an unexpectedly wide host and geographical range. An unnamed trypanosomatid isolated from rats in Egypt was found to belong to the genus Herpetomonas, so far associated with insect hosts only. It is closely related to Herpetomonas ztiplika, a parasite of a blood-sucking biting midge. Apparently several different trypanosomatid species can infect one insect species, as exemplified by Leptomonas sp. PL and Wallaceina sp. Wsd, which were isolated from different specimens of Salda littoralis on the same locality and day. However, since the same species of Leptomonas was obtained from insect hosts belonging to different genera, some insect trypanosomatids may have low host specificity. Our data revealed additional discrepancies between molecular phylogenetic data and cell morphology, rendering current trypanosomatid taxonomy unreliable.

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.

We describe the discovery and developmental features of a Helicosporidium sp. isolated from the black fly Simulium jonesi. Morphologically, the helicosporidia are characterized by a distinct cyst stage that encloses three ovoid cells and a single elongate filamentous cell. Bioassays have demonstrated that the cysts of this isolate infect various insect species, including the lepidopterans, Helicoverpa zea, Galleria mellonella, and Manduca sexta, and the dipterans, Musca domestica, Aedes taeniorhynchus, Anopheles albimanus, and An. quadrimaculatus. The cysts attach to the insect peritrophic matrix prior to dehiscence, which releases the filamentous cell and the three ovoid cells. The ovoid cells are short-lived in the insect gut with infection mediated by the penetration of the filamentous cell into the host. Furthermore, these filamentous cells are covered with projections that anchor them to the midgut lining. Unlike most entomopathogenic protozoa, this Helicosporidium sp. can be propagated in simple nutritional media under defined in vitro conditions, providing a system to conduct detailed analysis of the developmental biology of this poorly known taxon. The morphology and development of the in vitro produced cells are similar to that reported for the achorophyllic algae belonging to the genus Prototheca.

Molecular data have proved useful as an alternative to morphological data in showing the relationships of genera within the phylum Microsporidia, but until now have been available only for ribosomal genes. In previous studies protein-coding genes of microsporidia have been used only to assess their position in the evolution of eukaryotes. For the first time we report on the use of a protein-coding gene, the A-G region of the largest subunit of RNA polymerase II (RPB1) from 14 mainly polysporous species, to generate an alternative phylogeny for microsporidia. Using the amino acid sequences, the genera and species fell into the same main groupings as had been obtained with 16S rDNA sequences, but the RPB1 data provided better resolution within these groups. The results supported the pairings of Trachipleistophora hominis with Vavraia culicis and Pleistophora hippoglossoideos with Pleistophora typicalis. They also confirmed that the genus Pleistophora is not monophyletic and that it will be necessary to transfer Pleistophora ovariae and Pleistophora mirandellae into one or more other genera, as has already been effected for Pleistophora anguillarum.

The random amplification of polymorphic DNA was used for easy, quick and sensitive assessment of genetic polymorphism within Phytomonas to discriminate isolates and determine genetic relationships within the genus. We examined 48 Phytomonas spp., 31 isolates from plants and 17 from insects, from different geographic regions. Topology of the dendrogram based on randomly amplified polymorphic DNA fingerprints segregated the Phytomonas spp. into 5 main clusters, despite the high genetic variability within this genus. Similar clustering could also be obtained by both visual and cross-hybridization analysis of randomly amplified synapomorphic DNA fragments. There was some concordance between the genetic relationship of isolates and their plant tissue tropism. Moreover, Phytomonas spp. from plants and insects were grouped according to geographic origin, thus revealing a complex structure of this taxon comprising several clusters of very closely related organisms.

The phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of the lower termite Reticulitermes speratus was examined by a strategy that does not rely on cultivation. From mixed-population DNA obtained from the termite gut, small subunit (16S-like) ribosomal RNA sequences were directly amplified by the polymerase chain reaction method using primers specific for eukaryotes. Comparative sequence analysis of the clones revealed two kinds of sequences, one from the termite itself and the other from a symbiotic protist. A fluorescent-labeled oligonucleotide probe for the latter sequence was designed and used in whole-cell hybridization experiments to provide direct visual evidence that the sequence originated from a larger hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that this hypermastigote and trichomonads shared a recent common ancestry.

Flagellated protists of the Class Hypermastigida have previously been classified on morphology alone, since no molecular sequences have been available. We have isolated DNA from 350 cells of the hypermastigote Trichonympha, manually collected from the hindgut of Zootermopsis angusticollis, and used this DNA as template for polymerase chain reaction amplification of the small-subunit ribosomal RNA gene. The DNA sequence of the amplified fragment is closely related to that of a previously-unidentified gut symbiont from the termite Reticulitermes flavipes, and phylogenetic analysis places both sequences as a sister group to the known trichomonads, in agreement with the morphological classification.

The SSUrDNA and the ITS of different microsporidia from eight fishes, four insects and a shrimp were amplified and digested with restriction enzymes. The generated riboprints suggest a close evolutionary relationship between Glugea americanus and Spraguea lophii suggesting that Glugea americanus should be renamed Spraguea americanus and that the tissue infected and host origin should be considered of greater taxonomic importance for defining a genus than previously considered. Phylogenetic analysis of the riboprints demonstrates an unidentified microsporidium from a bumper fish (Chloroscombrus chrysurus) is related although not identical to Microgemma ovoidea, a parasite from red band fish. We were also able to distinguish between Glugea anomala and Glugea atherinae and Glugea stephani but were not able to differenciate among the latter two. Insects isolates, Nosema costelytrae, N. bombycis, N. trichoplusiae, Nosema sp. and a shrimp isolate, Agmasoma penaei, are not related to the fish isolates.

A trichomonad flagellate strain R1 was isolated from the hindgut contents of the termite Reticulitermes santonensis Feytaud. The flagellate was cultivated at 28 degrees C in anaerobic medium containing yeast extract, minerals and vitamins. The isolate fed on living bacteria. It showed the typical morphological and ultrastructural features of the trichomonads, closely resembling Trichomitus trypanoides. In order to determine its phylogenetic position the small subunit ribosomal DNA (SSU rDNA) of the flagellate was amplified in vitro using the polymerase chain reaction (PCR), cloned in a plasmid vector and sequenced. Comparison of the obtained sequence with so far available SSU rRNA/rDNA sequences showed strongest similarity (89%) to the sequence of Tritrichomonas foetus. The phylogenetic analysis with parsimony and distance matrix methods placed Trichomitus trypanoides strain R1 near by the root of the phylogenetically so far analyzed eukaryotic organisms. This confirms that termites harbour hindgut symbionts, which originate from very early evolved eukaryotes.