Important scientific discoveries have utilized the unique advantages of Tetrahymena thermophila as a research organism. Recently developed molecular genetic manipulations allow full exploitation of the many scientific dividends that would result from having its genome sequenced. As a typical ciliated protozoan, Tetrahymena exhibits “nuclear dimorphism”. It possesses two differentiated forms of its nuclear genome: a globally repressed, diploid germline or micronuclear genome, and a polyploid, site-specifically fragmented somatic or macronuclear genome. The macronuclear genome is, in effect, a natural, large-insert library of the micronuclear genome. This presentation describes how the gifts of nuclear dimorphism are being exploited in the experimental analysis of molecular and cell biology. Mechanisms present in humans that are either absent in other eukaryotic microbial model systems, or not as readily accessible in them as in Tetrahymena, are especially relevant. This presentation also reviews unique tools generated by nuclear dimorphism that are being used for genetically and physically mapping the Tetrahymena genome.

Dictyostelium discoideum is a useful model for molecular studies of cell biology and development. The 34-megabase Dictyostelium genome is currently being sequenced through the efforts of an international consortium. The genome is expected to encode 8–10,000 genes, including all those required for a free-living eukaryote capable of multicellular development. A complete description of the Dictyostelium genome will open the way toward the application of genome-based experimental approaches to studies of cell biology and development in this organism, and allow detailed physiological and evolutionary comparisons to other species.

Chlamydomonas reinhardtii has been the subject of genetic, biochemical, cytological, and molecular analyses for over 50 years. It is an ideal model system for the study of flagella and basal bodies as well as the study of photosynthesis and chloroplast biogenesis, cell-cell recognition and fusion, phototaxis, and secretion. It is clear that many of the genes identified in Chlamydomonas have homologs in land plants as well as animals. Thus, a genomic approach in Chlamydomonas will provide another important avenue for the understanding of important biological processes.

High resolution video-microscopy was used to observe grazing patterns of the heterotrophic nanoflagellates Cafeteria roenbergensis, Bodo saltans, Spumella sp., and Ochromonas sp. Spumella and Ochromonas enclose food particles with pseudopodia while Cafeteria and Bodo engulf particles by invagination of the cell surface. The following parameters of the feeding process were quantified: frequency of flagellar beating, speed of particles in different positions of the feeding current, food size selection, feeding rate, and the time budget for the handling of particles. The mean handling times differed between 94 s for Cafeteria and 4 s for Ochromonas for ingested particles. Handling times for ingested particles were significantly longer than for non-captured particles. Long handling times were calculated to be disadvantageous only for flagellates which propel a high water volume per hour (esp. Ochromonas) or live in a bacteria-rich environment. Our model calculations may provide a reasonable theoretical explanation for a concentration-dependent behavioural variability of the feeding strategy of different heterotrophic nanoflagellates (HNF) species.

To complete our investigations on the oriented behavioral response of isotropically cooled, inert populations of Oxytricha bifaria to a warm thermal gradient, their physiological potentialities under cold microgradient conditions arising at 8.5 °C were studied. We monitored the behavior of the experimental populations, both at the level of the passing cold wave front, and afterwards when the thermal gradient stabilized, evaluating (i) their distribution in general, (ii) their relative centroids, (iii) the percentage of both backward creeping and immobile ciliates, and (iv) the numerical indices and rates of their creeping tracks. At the arrival of the cold wave front, the oxytrichas react immediately to the thermal stimulus, creep backwards at very high velocity along uninterrupted linear tracks, and thus move away from the cooling source. No specific behavioral response was ever observed in the static microgradient conditions. At 8.5 °C, despite their inertness, the ciliates are still able to behave adaptively, reacting immediately and orientatedly, once a directional factor (the cold-repelling thermal gradient) arises in an isotropic environment. This is similar to their behavior in the symmetric warm attracting thermal gradient.

Multiple copies of a gene may lead to difficulty in the interpretation of typing results because polymorphism of the copies may wrongly lead to the conclusion that different types are present in a specimen. To determine the copy number per genome of the nuclear rDNA and beta-tubulin genes analyzed for the typing of Pneumocystis carinii f. sp. hominis, we developed a strategy based on the use of the same multicompetitor molecule in two different quantitative-competitive PCRs, one for the gene under study and the other for a reference single copy gene, allowing direct comparison of the results of both PCRs. Control experiments showed that the strategy was sensitive enough to detect duplication of a gene. The copy number of the nuclear rDNA operon was determined by amplification of the intron of the 26S rDNA gene and that of the beta-tubulin by amplification of the region surrounding the intron no. 6. The method was first tested on P. c. carinii, the special form commonly infecting rats. Pneumocystis c. carinii was found to contain a single copy of the rDNA operon. The method was then applied to P. c. hominis. The results confirmed that P. c. hominis genome contains a single copy of the nuclear rDNA and beta-tubulin genes.

The neutral lipids and their fatty acids and the sterol fractions of the marine ciliated protozoon, Parauronema acutum, were characterized. The neutral lipids consisted of triglycerides (30%), sterols (29%), free fatty acids (24%), steryl esters (9%), and diglycerides (8%) and small amounts of fatty alcohols. The fatty acid profiles of these lipids were very similar although quantitative differences were detected. Saturated fatty acids, primarily 14:0, 16:0, and 18:0 constituted 20–30% of the total. Unsaturated fatty acids containing one to three double bonds, primarily 18:1(9), 18:2 (9,12), 18:3 (9, 12, 15) and 20:3 (11, 14, 17), constituted 35–50% of the total. Highly unsaturated fatty acids, 18:4 (6, 9, 12, 15), 20:5 (5, 8, 11, 14, 17) and 22:6 (4, 7, 10, 16, 19), constituted 16–25% of the total. The fatty alcohols consisted of 14:0 (2%), 16:0 (66%), 18:0 (3%), 20:0 (8%), and 22:0 (21%). The sterols of Parauronema acutum consisted of cholesterol (53%), campesterol (32%), desmosterol (7%), and β-sitosterol (8%).

Nucleospora salmonis is an intranuclear microsporidian associated with a proliferative disorder of the lymphoid cells of captive salmonid fish in the northwestern and northeastern regions of North America, in France, and in Chile. Newer diagnostic approaches have used the polymerase chain reaction (PCR) to detect the parasite in fish tissues. The target sequences for these assays lie in the small subunit ribosomal RNA (ssu rRNA) gene or internal transcribed spacer (ITS) as determined from N. salmonis from chinook salmon (Oncorhynchus tshawytscha) from the Pacific Northwest of North America. The lack of sequence data on parasites from diverse geographic origins and hosts led us to compare several isolates of N. salmonis. There was a high degree of similarity in the ssu rDNA sequences (> 98%) among all the isolates of N. salmonis examined, regardless of host or geographic origin. The greatest sequence differences were found between isolates from the Pacific regions of America. Isolates from Chile shared sequences with one or both geographic groups from North America. A similar distribution of sequence types was observed when ITS-1 sequences of selected isolates were analyzed. Sequence data from two N. salmonis-like isolates from marine non-salmonid fish showed one closely related and the second less closely related to N. salmonis isolates from salmonid fish. These results provide evidence for a homogeneous group of aquatic members of the genus Nucleospora found among salmonid fish (N. salmonis) that can be detected using diagnostic PCR assays with ssu rDNA target sequences. The presence of parasites related to N. salmonis among marine fish suggests a potentially broad host and geographic distribution of members of the family Enterocytozoonidae.

The southern green stink bug, Nezara viridula (L.), and certain of its host plants were examined to determine the prevalence and biological characteristics of an intestinal trypanosomatid. Promastigotes with short (≤ 17.5 μm excluding flagellum) and long forms (≥ 25.0 μm) usually infected < 50% of the bugs before August and > 50% (maximum 95%) during August–October, but prevalence was not host-density dependent. The flagellate was detected in adults and in all nymphal instars, at all sampling sites where at least 10 bugs were captured, and in bugs from all host plants sampled (soybean, red clover, vetch). Of bugs with flagellates, 27% were heavily infected (> 20 flagellates per 160× microscope field). Weights of infected and uninfected adults did not differ. Live flagellates were detected in bug feces and in one stem of red clover. When bugs were fed soybean pods, tomatoes, or snap beans in the laboratory, only once were flagellates detected in plant tissue (snap beans). The flagellate was cultured in modified Medium 199. This flagellate is prevalent in N. viridula populations in Louisiana and apparently does not cause significant pathological effects in N. viridula or its host plants, including soybean.

Distribution of immune cell populations was studied in a C3H/HeJ mouse model of intestinal amebiasis from 5 to 60 days post-inoculation with Entamoeba histolytica, using immunoperoxidase techniques. At various time intervals, the ceca from mice were fixed in 10% formalin, dehydrated, embedded and sectioned at 5 μm. Sections were incubated with conjugated peroxidase-labelled antibodies to mouse IgA, IgM, and IgG. Color was developed with 3, 3′-diaminobenzidine tetrahydrochloride (DAB)/H₂O₂ solution. CD3, CD4, and CD8 cells, as well as neutrophils were detected by reacting with biotin-conjugated anti-mouse CD3, CD4, CD8, and CD11 monoclonal antibodies, followed by their incubation with avidin-peroxidase and color development with DAB/H₂O₂ solution. Erythrocin B and toluidine blue were used to stain eosinophils and Mast cells, respectively. It was observed that the IgA plasma cell was the dominating immune cell present in the mucosa, although eosinophils, neutrophils, CD3 , CD4 , CD8 , IgM , IgG cells and Mast cells were also seen. Results of this study suggest that infiltration of immune cells at the mucosal surface during intestinal amebiasis might be important in the defense against this parasite.

Eighteen strains of flagellated protists representing nine species were isolated and cultured from four deep-sea hydrothermal vents: Juan de Fuca Ridge (2,200 m), Guaymas Basin (2,000 m), 21° N (2,550 m) and 9° N (2,000 m). Light and electron microscopy were used to identify flagellates to genus and, when possible, species. The small subunit ribosomal RNA genes of each vent species and related strains from shallow-waters and the American Type Culture Collection were sequenced then used for comparative analysis with database sequences to place taxa in an rDNA tree. The hydrothermal vent flagellates belonged to six different taxonomic orders: the Ancyromonadida, Bicosoecida, Cercomonadida, Choanoflagellida, Chrysomonadida, and Kinetoplastida. Comparative analysis of vent isolate and database sequences resolved systematic placement of some well-known species with previously uncertain taxonomic affinities, such as Ancyromonas sigmoides, Caecitellus parvulus, and Massisteria marina. Many of these vent isolates are ubiquitous members of marine, freshwater, and terrestrial ecosystems worldwide, suggesting a global distribution of these flagellate species.

In Tetrahymena thermophila mating type alleles specify temperature sensitive frequency distributions of multiple mating types. A-like alleles specify mating types I, II, III, V and VI, whereas B-like alleles specify mating types II through VII. We have characterized the mating type distributions specified by several A- and B-like genotypes segregated by genomic exclusion from cells isolated from a pond in northwestern Pennsylvania. The B-like genotypes are alike in specifying very low frequencies of mating type III, but differ with respect to the frequencies of other mating types, particularly II and VII. An A-like genotype specifies a high frequency of mating type III and is unstable in successive generations for the expression of mating type II, suggesting a possible modifier. Inter se crosses performed at 18 °C, 28 °C and 34 °C showed that each genotype specifies a frequency distribution that is uniquely affected by temperature. No mating type was affected the same way by temperature in all genotypes. In A/B heterozygotes, the B-like genotype exhibited partial dominance. The genotypes described here differ significantly from previously described genotypes from the same pond, indicating that there are numerous mating type alleles. For frequency-dependent selection to equalize mating type frequencies, it must act not only on complex multiple alleles but also on the response of mating type alleles to temperature.

Peniculine ciliates have been recognized as a distinct higher taxon of ciliates for almost 50 years. However, phylogenetic relationships within the Subclass Peniculia are still unsettled. To contribute to our understanding of their phylogeny and provide evidence for the position of Urocentrum turbo, we sequenced its small subunit (SS) rRNA gene and the SSrRNA genes from Lembadion bullinum, Frontonia sp., Paramecium caudatum, Paramecium multimicronucleatum, Paramecium putrinum, and Paramecium woodruffi. Urocentrum turbo was the only one of these species not to exhibit a shortened Helix E10_1, which we conclude characterizes the “higher” peniculines. Except for U. turbo, the peniculines are strongly supported as a monophyletic clade with Lembadion, Frontonia, and Paramecium species forming separate and strongly supported clades by bootstrap analysis using distance matrix, maximum parsimony, and maximum likelihood methods. Urocentrum turbo is associated with different lineages, depending upon the analysis used. The Paramecium species form at least four clades with the Paramecium aurelia subgroup being the most derived. We conclude that the Subclass Peniculia should be divided into two orders, the Order Urocentrida and Order Peniculida, with the latter order having two suborders, the Suborder Frontoniina and Peniculina. We place U. turbo with the peniculines because of shared morphological and stomatogenetic features.