During macronuclear development in the ciliate Euplotes crassus, micronuclear-derived chromosomes undergo a series of rearrangements that include polytenization, DNA splicing, chromosome fragmentation, and telomere addition and processing. Although cis-acting signals that may function in the regulation of these events have been characterized, the proteins that mediate these events have not yet been identified. To identify development-specific factors that may be involved in DNA rearrangement, we previously isolated clones of a number of genes that are expressed only during early macronuclear development. Here, we report the genomic and cDNA sequences of one of these genes, conZA8. The analysis indicates that the conZA8 gene encodes a novel, 468-amino acid, proline-rich protein. Antibodies were raised against both a recombinant form of the conZA8 protein and an internal peptide. Immunoblotting and immunofluorescence analyses indicated that the conZA8 protein is highly abundant, expressed only during the polytene chromosome stage of macronuclear development, and localized to the developing macronucleus. Possible functions of the conZA8 protein are discussed.

Internal transcribed spacers (ITS) and the 5.8S ribosomal gene of 21 Naegleria fowleri strains and eight other species including Naegleria gruberi were sequenced. The results showed that this region can help differentiate between and within species. The phylogeny of Naegleria spp. deduced from the ITS and the 5.8S gene produced four major lineages, fowleri-lovaniensis, galeacystis-italica-clarki-gruberi-australiensis, andersoni-jamiesoni, and pussardi, that fit perfectly with those inferred from the 18S rRNA gene analysis. The N. gruberi isolate, NG260, was closely related to Naegleria pussardi. The other N. gruberi isolates branched together with Naegleria australiensis in another lineage. The ITS and 5.8S results for N. fowleri were congruent with those previously deduced by RAPD analysis. The phylogenetic analysis inferred from ITS and RAPD data revealed two major groups. The French Cattenom and Chooz and South Pacific strains constituted the first group. The second group encompassed the strains corresponding to the Euro-American and Widespread RAPD variants and shared the same substitution in the 5.8S gene. In addition, it was possible to define species specific primers in ITS regions to rapidly identify N. fowleri.

Within the frame of the de novo formation of Platelet-Activating Factor in Tetrahymena, the occurrence as well as the properties of a lipid phosphate phosphohydrolase activity catalyzing the dephosphorylation of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphate was investigated. The activity was distributed in all the membrane fractions of the cell and in the cytosol. It showed preference for acyl-acetyl-sn-glycero-phosphate as well, and at a much lower level, for dipalmitoyl-glycero-phosphate. Mg² and Ca² caused a dose-dependent inhibition, while F⁻, EDTA and EGTA had no effect. The enzymic activity was linear for at least up to 60 min incubation time and up to 150 μg protein. Microsomal activity exhibited two optimal pH areas, around 7.0 and 9.0, while mitochondrial activity showed one peak, at pH 7.0. Acyl-GP, acyl-acetyl-GP and alkyl-GP could replace alkyl-acetyl-GP in significant rates, while dipalmitoyl-GP, β-GP, fructose-6-GP, p-nitrophenylphosphate, creatine phosphate or ATP had no effect. Side phospholipase A² and C activities were also detected. Taking into account the presence of PAF and alkylacetylglycerol in the protozoan as well as the presence of a dithiothreitol-insensitive CDP-choline:cholinephosphotransferase activity that converts alkylacetylglycerol to PAF, we suggest that the present phosphohydrolase activity may be involved in the de novo production of PAF within Tetrahymena.

Anti-centrin monoclonal antibodies 20H5 and 11B2 produced against Chlamydomonas centrin decorated the group of basal bodies as well as very closely attached structures in all trichomonads studied and in the devescovinids Foaina and Devescovina. Moreover, these antibodies decorated the undulating membrane in Trichomonas vaginalis, Trichomitus batrachorum, and Tritrichomonas foetus, and the cresta in Foaina. Centrin was not demonstrated in the dividing spindle and paradesmosis. Immunogold labeling, both in pre- and post-embedding, confirmed that centrin is associated with the basal body cylinder and is a component of the nine anchoring arms between the terminal plate of flagellar bases and the plasma-membrane. Centrin is also associated with the hook-shaped fibers attached to basal bodies (F1, F3), the X-fiber, and along sigmoid fibers (F2) at the pelta-axostyle junction, which is the microtubule organizing center for pelta-axostyle microtubules. There was no labeling on the striated costa and parabasal fibers nor on microtubular pelta-axostyle, but the fibrous structure inside the undulating membrane was labeled in T. vaginalis. Two proteins of 22–20 kDa corresponding to the centrin molecular mass were recognized by immunoblotting using these antibodies in the three trichomonad species examined.

By screening a T. vaginalis cDNA library with 20H5 antibody, two genes encoding identical protein sequences were found. The sequence comprises the 4 typical EF-hand Ca -binding domains present in every known centrin. Trichomonad centrin is closer to the green algal cluster (70% identity) than to the yeast Cdc31 cluster (55% identity) or the Alveolata cluster (46% identity).

Conjugant pairs of Tetrahymena thermophila were mechanically separated by vigorous pipetting at the early stages of meiotic prophase. The complete sequence of conjugational nuclear events including the appearance of pronuclei, development of the new macronuclei (postzygotic development), and resorption of the old macronuclei was observed in the separated cells, without pronuclear exchange. The pronuclei in the separated cells were recognised by the presence of components of the extranuclear cytoskeleton, which were labelled with anti-tubulin and anti-fenestrin antibodies in the same way as in undisturbed conjugants. The apical region of the separated conjugants (the post-junction area), corresponding to the junction area of conjugants was labelled with anti-fenestrin antibody and maintained the properties required for the nuclear development. The results of the genetic study were consistent with a hypothesis that cytogamy (pronuclear fusion) was induced in the separated conjugants. Therefore, the lasting cell contact is not necessary for the successful completion of conjugational nuclear events.

The abundance, sizes, and when appropriate, diversity of gymnamoebae were documented at approximately monthly intervals for four years (1995–1998) at a grassy, terrestrial site slightly upslope from a freshwater pond. Soil samples were analyzed for viable gymnamoebae using a standard laboratory culturing protocol. The mean density of gymnamoebae based on the total data set was ca.1,600/g (s.e. ± 190). Minimum densities of gymnamoebae (156/g) occurred in January 1995, and a maximum for the sampling period (5,838/g) occurred in July 1997, when a rainy period followed an extended period of drought. Among the environmental variables monitored (precipitation, soil moisture, organic content, and temperature) only precipitation correlated significantly with abundance of gymnamoebae (r = 0.34, p = 0.02). During the mild, moist El Niño winter of 1997–1998, a larger than usual number of gymnamoebae was recorded at the site (∼3,800/g) compared to a mean density of ∼900/g for comparable periods in preceding years. The mean sizes were also larger. Since gymnamoebae are increasingly recognized as major members of soil microbial communities enhancing soil fertility through nutrient mineralization, it is important to document environmental variables that influence their abundance and activity in terrestrial ecosystems.

The tracks of normal organisms of Oxytricha bifaria and of stage IA, IB, II, III, IV and V doublets were studied to test the hypothesis that the doublet might function as a dispersal form. Stage IA, the only stage to swim, swims straight with only rare interruptions; its rate of mobility (R_mo = 443 μ/s) is roughly twice that of singlets (R_mo = 218 μ/s). Stage IA doublets swim in three-dimensional movement which enables them to be carried away by water currents. The other stages seem to represent passage back towards the normal singlet form. The ethological evidence reported here together with other results already published supports the working hypothesis that the doublet of O. bifaria is a dispersal form suggests that the doublet might well represent a special fourth differentiation state of this species in addition to pairs, giants, and cysts.

Six genera of rotifers including Philodina, Monostyla, Epiphanes, Euchlanis, Brachionus, and Asplanchna were exposed to oocysts of Cryptosporidium parvum cleaned of fecal debris. Unstained oocysts and those stained with fluorescein-conjugated monoclonal antibody were added to suspensions of viable rotifers and were examined by phase-contrast, differential interference contrast, and fluorescence microscopy. Rotifers of all six genera were observed ingesting oocysts. A maximum of 25 oocysts was observed in the stomachs of Euchlanis and Brachionus. Euchlanis and Epiphanes were observed excreting boluses containing up to eight oocysts. It was not determined whether rotifers digested or otherwise rendered oocysts nonviable.

Genetic and biochemical characterization of microbes often requires the use of clonal cultures. A method to clone the oyster parasite Perkinsus marinus is described. Individual cells are isolated via micromanipulation and maintained above an actively proliferating “feeder layer” of P. marinus on a 0.45-μm membrane. Extracellular products released from the proliferating feeder layer can diffuse across the membrane and bathe the isolated cell, stimulating it to proliferate. The method is relatively simple and should be applicable to most protists that can be cultured in the laboratory.

The three taxa emerging at the base of the eukaryotic ribosomal RNA phylogenetic tree (Diplomonadida, Microspora, and Parabasalia) include a wide array of parasitic species, and some free-living organisms that appear to be derived from a parasitic ancestry. The basal position of these taxa, which lack mitochondria, has recently been questioned. I sequenced most of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis and a secondary structure model was reconstructed for the SSU rRNA. I conducted a RASA matrix analysis to identify, independently from tree reconstruction, putative long branch attraction effects in the data matrix. The results show that each of the basal clades and the euglenozoan clade act, indeed, as long branches and may have been engaged in a process of accelerated rate of evolution. A nucleotide signature analysis was conducted in the conserved regions for positions defining the three great domains of life (Eubacteria, Archea, and Eukaryota). For the three basal taxa, this analysis showed the presence of a significant number of different non-eukaryotic nucleotides. A precise study of the nature and location of these nucleotides led to conclusions supporting the results of the RASA analysis. Altogether, these findings suggest that the basal placement of these taxa in the SSU ribosomal RNA phylogenetic tree is artifactual, and flawed by long branch attraction effects.

Rumen ciliate species composition was surveyed in domestic yaks kept in Tibet, Sichuan, and Inner Mongolia, China. Twelve genera including 36 species with 18 formae were identified. The species compositions were slightly different among the three areas: yaks in Tibet had the simplest fauna, in contrast, the fauna of yaks in Inner Mongolia were the most abundant and similar to those found in the cattle kept in the same area. This suggests that the rumen ciliate composition of yaks is affected by that of cattle kept together or in proximity. A new species belonging to the genus Entodinium, Entodinium monuo n. sp., was recognized from the yaks in all areas. This new species was common in the yaks but was not detected in the cattle fed near yaks in Inner Mongolia. There was a similar generic ciliate composition in yaks kept in respective areas. Entodinium was the most predominate ciliate (51.9–61.0%) with total ciliate densities estimated as 10⁵/ml per yak.