Chlamydomonas reinhardtii is a unicellular green alga that has been used as a model organism for the study of flagella and basal bodies as well as photosynthesis. This report analyzes finished genomic DNA sequence for 0.5% of the nuclear genome. We have used three gene prediction programs as well as EST and protein homology data to estimate the total number of genes in Chlamydomonas to be between 12,000 and 16,400. Chlamydomonas appears to have many more genes than any other unicellular organism sequenced to date. Twenty-seven percent of the predicted genes have significant identity to both ESTs and to known proteins in other organisms, 32% of the predicted genes have significant identity to ESTs alone, and 14% have significant similarity to known proteins in other organisms. For gene prediction in Chlamydomonas, GreenGenie appeared to have the highest sensitivity and specificity at the exon level, scoring 71% and 82%, respectively. Two new alternative splicing events were predicted by aligning Chlamydomonas ESTs to the genomic sequence. Finally recombination differs between the two sequenced contigs. The 350-Kb of the Linkage group III contig is devoid of recombination, while the Linkage group I contig is 30 map units long over 33-kb.

Spores of four species of microsporidia isolated from humans were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and specific biomarkers were found for each. The microsporidia analyzed included three species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis and the fourth organism is the recently described Brachiola algerae. Whole spores, spore shells, and soluble fractions were applied directly to the MALDI target without further purification steps. MALDI-TOF MS analysis of both whole spores and soluble fractions of the four isolates revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 2,000–8,000 Da. Statistical analysis of the averaged centroided masses uncovered two distinct sets of unique peptides or biomarkers, one originated from whole spores and the other from soluble fractions, that can differentiate the four microsporidian species studied. MALDI-TOF MS analysis of whole organisms is a rapid, sensitive, and specific option to characterize microsporidian isolates and has the potential for several applications in parasitology.

The inositol isomer composition of phosphoinositides, polyphosphoinositols, phosphatidylinositol-linked glycans, and glycosyl phosphatidylinositol-anchored proteins of logarithmic phase Tetrahymena vorax was determined by GC-MS analysis of trimethylsilylimadazole derivatives. The most abundant inositol found was the myo-isomer; however, appreciable percentages of scyllo-inositol were present in the free inositol pool, phosphatidylinositol-linked glycan fraction, and glycosyl phosphatidylinositol-anchored protein fraction. Trace quantities of chiro- and neo-inositols also were present.

Microsporidia are obligate intracellular pathogens of invertebrate and vertebrate animals. Most human infections are caused by Enterocytozoon bieneusi or Encephalitozoon intestinalis, and result in chronic diarrhea. In order to determine the signals involved in microsporidial spore activation and invasion, kinetics of in vitro E. intestinalis replication were defined using real-time quantitative PCR. Segments of small subunit ribosomal RNA and polar tube protein 2 genes of E. intestinalis were used to quantify parasite gene copy number following infection in murine colon carcinoma cells. Parasite DNA was detectable in small but significant amounts within host cells as early as 4 h postinoculation, genome replication was completed by 36 h, and parasite progeny were released into the supernatant beginning 72 h postinoculation. Heat-treating spores did not prevent transfer of parasite DNA into cells, but did inhibit parasite replication. Treating cell cultures with albendazole suppressed but did not completely inhibit parasite replication. These results confirm observations that E. intestinalis completes its life cycle within the turnover time of its target host cells; invasion into susceptible host cells occurs independently of spore viability; and real-time quantitative PCR is a sensitive and reproducible method with which to monitor microsporidial infection under varying treatments or conditions.

We combined behavioral, morphological (live, stained, scanning and transmission electron-microscope), and molecular data to redescribe two common, intertidal oligotrich ciliates, Novistrombidium testaceum and Strombidium inclinatum. Both species were collected from a rocky shore near Leghorn, Ligurian Sea. A literature review revealed four morphotypes of Novistrombidium testaceum that differ in subtle ways, including oral morphology. These differences may be diagnostic, but we do not consider them sufficient to distinguish different taxa. Although other studies have synonymised Strombidium inclinatum and S. sulcatum, based on oral structures, size, and nuclear structure, there are morphological distinctions between them. In particular, the present study supports a lack of anterior protuberance in both live and preserved S. inclinatum, while S. sulcatum possesses a protuberance. The 18S rDNA molecular data, in accordance with morphological and ultrastructural observations, indicate that the Strombidiida (Oligotrichia) constitute a well-supported clade. The separation of the genera within this clade, even between Novistrombidium and Strombidium, remains unresolved, and the analysis of more species is required. Finally, we recommend that when possible, ecologists, morphological taxonomists, and molecular biologists combine their expertise to provide comprehensive taxonomic descriptions.

Oxymonads are a morphologically well-characterized and highly diverse lineage of protists. They are, however, under sampled at a molecular level. It has recently been demonstrated that a genus of oxymonads, Pyrsonympha, is phylogenetically related to the excavate taxon Trimastix. Here, we addressed issues of internal oxymonad evolution. Pyrsonympha and Dinenympha are shown, by fluorescent in situ hybridization and phylogenetic evidence, to be separate genera and not morphotypes of the same organism. We demonstrated that three genera of oxymonads, Dinenympha, Pyrsonympha, and Oxymonas are each monophyletic and together form a clade which excludes other known eukaryotes. We have presented a taxonomic scheme of oxymonads taking into account their sisterhood with Trimastix and speculated on morphological evolution of oxymonads, particularly of their attachment apparatuses. Our biogeographical analysis with Japanese and Canadian Pyrsonympha and Dinenympha suggests that these genera diverged before the separation of termites that inhabit Eastern Asia and Western North America.

A comparison of random amplified polymorphic DNA (RAPD) was used to investigate genetic polymorphisms among 25 isolates of Giardia intestinalis and to assess the utility of RAPD for subtype detection and genealogical analysis. Using data obtained for six human and 19 animal-derived isolates in polymerase chain reactions using 13 different primers, phylogenetic trees were constructed and bootstrap values computed by the program FreeTree. Three major clades were distinguished, corresponding to previously defined genetic assemblages A, B, and E. The purported specificity of assemblage E genotypes for artiodactyl hosts was supported. Assemblages A and B showed wide host spectra, including human and animal hosts. No correlation was found between the genotype of analyzed isolates and the presence or absence of the double-stranded RNA Giardiavirus. The results indicate that RAPD data provide reliable genetic information that can be used for both “fingerprinting” and genealogical purposes.

A new epibiontic ciliate of the genus Metacystis is described on the seagrass Thalassia testudinum of the coral reef lagoons of Veracruz, Mexico. The ciliate was studied in living and stained specimens and under the scanning electron microscope. The cell body (10–35 × 10–18 μm in vivo) is transversely annulated (4–6 rings). The somatic ciliature consists of 22–30 longitudinal kineties, and patterned as 5–7 transverse kineties. The circumoral kinety is composed of kinetosomes closely spaced. The macronucleus diameter measures about 3–7 μm. The lorica (18–61 × 11–26 μm) has the posterior end round to conical or irregular with mucoid filaments. This prostomatid colonizes both natural and artificial substrates placed in an aquarium. Metacystis borrori n. sp. is a species that forms part of the ciliate community on Thalassia testudinum with a temperature range of 21–26 °C and a salinity of 32–40‰.

The amitochondriate protistan parasite Entamoeba histolytica has lost most mitochondrial functions secondarily but has retained a reduced organelle of mitochondrial origin, the mitosome. We here investigate the presence, origins, and expression in other species of Entamoeba of three genes of mitochondrial origin—pyridine nucleotide transhydrogenase and the mitochondrial-type chaperonins cpn60 and hsp70. The genes appear to be present in all species and specifically related, confirming that the E. histolytica mitosomal genes were not acquired recently by lateral transfer from another organism. Detection of expression was not possible in all cases under the culture conditions used, but several genes were induced during recovery from exposure to a heat shock. This includes the transhydrogenase, which to our knowledge has not been shown previously to be a heat-shock protein.

Fish species around the world are parasitized by myxozoans of the genus Kudoa, several of which infect and cause damage of commercial importance. In particular, Kudoa thyrsites and Kudoa amamiensis infect certain cultured fish species causing damage to muscle tissue, making the fish unmarketable. Kudoa thyrsites has a broad host and geographic range infecting over 35 different fish species worldwide, while K. amamiensis has only been reported from a few species in Japanese waters. Through morphological and molecular analyses we have confirmed the presence of both of these parasites in eastern Australian waters. In addition, a novel Kudoa species was identified, having stellate spores, with one polar capsule larger than the other three. The SSU rDNA sequence of this parasite was 1.5% different from K. thyrsites and is an outlier from K. thyrsites representatives in a phylogenetic analysis. Furthermore, the spores of this parasite are distinctly smaller than those of K. thyrsites, and thus it is described as Kudoa minithyrsites n. sp. Although the potential effects of K. minithyrsites n. sp. on its fish hosts are unknown, both K. thyrsites and K. amamiensis are associated with flesh quality problems in some cultured species and may be potential threats to an expanding aquaculture industry in Australia.

A study was carried out to determine if the protozoal fauna of indigenous African wild ruminants was different from that found in their domestic counterparts and if the animal's diet influenced the number and types of protozoa. Samples of rumen contents were collected in 1997 and 2001 from various indigenous African wild ruminants in Kenya. All three ruminant feeding types were sampled: browsers or concentrate selectors (giraffe and Guenther's dik-dik); intermediate or adaptable mixed feeders (impala, Thomson's gazelle, Grant's gazelle and eland); grass or roughage eaters (hartebeest and wildebeest). Total concentration of ciliate protozoa and percentage generic distribution were determined. In general, protozoal concentrations were higher in concentrate selectors, followed by the intermediate or opportunistic mixed feeders and lowest in the grass and roughage eaters. Both Thomson's and Grant's gazelle were protozoa-free in the 2001 samples. Entodinium percentages were considerably higher in concentrate selectors and intermediate mixed feeders, compared to roughage eaters. Two genera of protozoa previously found in only a few African ruminants, Epiplastron and Opisthotrichum, were observed in several additional animal species and represent new host records. A difference was noted in the protozoal species composition of the indigenous wild ruminants from that previously observed in African domestic ruminants.

Phylogenetic analysis of small-subunit ribosomal RNA gene sequences for gymnamoebae of the families Vexilliferidae, Paramoebidae, and Vannellidae identified two distinct lineages that are supported by gross morphological characters. This analysis indicates that paramoebids and vexilliferids are part of one lineage and that vannellids belong to another. A shared morphological character unique to the paramoebid/vexilliferid lineage members is the presence of dactylopodiate subpseudopodia. However, cell surface structures, normally used for taxonomic discrimination, range from simple hair-like filaments without any apparent organization (Neoparamoeba), to hexagonal glycostyles (Vexillifera) or more elaborate surface scales (Korotnevella). Taxa within the vannellid lineage all lack subpseudopodia and appear flabellate, spatulate or linguiform while in locomotion. Cell surface structures of taxa within the vannellid lineage range from filaments organized into hexagonal arrays (Lingulamoeba, Platyamoeba) to pentagonal glycostyles (Clydonella, Vannella). Vannellid lineage members of the genera Clydonella and Lingulamoeba were studied at the level of electron microscopy. Unique cell surface features validate these as genera distinct from Vannella and Platyamoeba. Genetic and ultrastructural data are used to discuss the phylogenetic interrelationships for the taxa studied.