Laboea strobila Lohmann, 1908 is a conspicuous oligotrich ciliate in the marine plankton. In order to compare different populations, the morphology of specimens from the Mediterranean Sea, North Sea, and Irish Sea was investigated using live observation, protargol impregnation, and scanning electron microscopy. Furthermore, the PCR-amplified products of the SSrRNA gene from a monoclonal culture of L. strobila from the Mediterranean Sea were sequenced and aligned with sequences of other oligotrichs, including a population of L. strobila from the Atlantic coast of the USA. Finally, the data from the ecological literature were summarized and the cultivation methods were described. The SSrRNA gene sequences of the two distantly located L. strobila populations from the North Atlantic are identical. Likewise, the morphometrics of most populations so far investigated after protargol impregnation (i.e. from the North Atlantic) do not show obvious differences. In all computed phylogenetic trees, L. strobila groups with Strombidium species, forming a monophyletic taxon corresponding to the subclass Oligotrichia. These results are corroborated by the ontogenetic comparison. Since no type species was fixed for Laboea Lohmann, 1908, L. strobila was designated in the present paper.

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.

Streblomastix strix is an enigmatic oxymonad found exclusively in the hindgut of the damp-wood termite Zootermopsis. Streblomastix has a number of unusual morphological characters and forms a complex but poorly understood symbiosis with epibiotic bacteria. Here we described the ultrastructure of S. strix, with emphasis on the axial cytoskeleton and cell-cell associations, in its normal state and when treated with antibiotics. In untreated cells, epibiotic bacteria were orderly arranged end-to-end on six or seven longitudinal vanes, giving S. strix a stellate appearance in transverse section. The epibiotic bacteria were unusually long bacilli of at least three different morphotypes. Bacteria adhered to the oxymonad host by distinct cell-cell junctions that protruded between the poles of adjacent epibiotic bacteria. Treating termites with the antibiotic carbenicillin led to the loss of most (but not all) of the bacteria and the transformation of S. strix from a long slender cell to a teardrop-shaped cell, where the axostyle was compacted and became bifurcated near the posterior end.

We have used small subunit rRNA gene sequences to determine the phylogenetic relationships of species in three genera of endosymbiotic ciliates. We have confirmed that the astome Anoplophrya marylandensis is related to ciliates in the Class Oligohymenophorea, supporting the view that astomes are derived from hymenostome-like ancestors. We confirmed that Plagiotoma lumbrici, formerly considered to be a heterotrich, is a stichotrich spirotrich ciliate most closely related to Paraurostyla weissei in this analysis. Thus, the somatic polykinetids of Plagiotoma can be concluded to be cirri. We report the details of our isolation of Nyctotheroides deslierresae and Nyctotheroides parvus and confirm previous reports that these clevelandellids are related to the metopid and caenomorphid ciliates, now placed in the Class Armophorea.

The transport characteristics of (1D)chiro-inositol by the ciliate Tetrahymena were examined in competition studies employing [³H](1d)chiro-inositol. (1d)chiro-Inositol transport was competed by unlabeled (1d)chiro-inositol, myo-inositol, scyllo-inositol, and d-glucose in a concentration-dependent manner. Conversely, (1d)chiro-inositol competed for [³H]myo- and [³H]scyllo-inositol transport. Lineweaver-Burke analysis of the competition data indicated a K_m of 10.3 mM and a B_max of 4.7 nmol/min/mg for (1d)chiro-inositol. Transport of (1d)chiro-inositol was inhibited by cytochalasin B, an inhibitor of facilitated glucose transporters, and phlorizin, an inhibitor of sodium-dependent transporters. Removal of sodium from the radiolabeling buffer also inhibited uptake. The presence of 0.64 mM calcium or magnesium ions exerted negligible effects on transport, although potassium was inhibitory. [³H](1d)chiro-Inositol was shown to be incorporated into Tetrahymena phosphoinositides.

Sequence analysis of genomic DNA from the protozoan parasite Perkinsus marinus at two loci revealed genetic polymorphisms within and among different cultured isolates. Genomic DNA from 12 Perkinsus marinus isolates was amplified at the internal transcribed spacer region and at an anonymous locus previously identified to contain polymorphisms by restriction fragment length polymorphism analysis. Fourteen polymorphic nucleotide positions were identified at the internal transcribed spacer region; eight in internal transcribed spacer 1 and six in internal transcribed spacer 2. Thirteen polymorphic nucleotide sites were identified within the anonymous locus. In some instances, more than three different sequences were observed at both the internal transcribed spacer region and at the anonymous locus from a single clonal isolate, suggesting the possibility of recombination in cultured cells and/or strand jumping during the polymerase chain reaction. Intra-isolate sequence variation (3.46% for the anonymous locus and 3.08% for internal transcribed spacer 1) was in several cases as high as inter-isolate sequence variation, even in one isolate where recombination was not evident. High intra- and inter-isolate variation detected at both loci demonstrates the importance of determining the genetic variation of each locus prior to development of sequence-based molecular diagnostics.

The resistance of Euglena (E.) gracilis to ionizing radiation was investigated using seven kinds of ion beams each with different energy characteristics. The minimum effective dose of the most lethal ion beams was 40 Gy. Given its substantially high resistance to heavy ion beams, E. gracilis possesses great potential in acting as an effective support system to produce food and regenerate oxygen in a space station. The lethal effect of ionizing radiation was dependent on the linear energy transfer value of the heavy ion beams, and reached a maximum at 196 keV/μm. This value was different from those obtained by previous irradiation experiments using mammalian and plant cells, suggesting that the radiation response of E. gracilis is distinct from that of mammalian and plant cells.

A “universal non-metazoan” polymerase chain reaction (UNonMet-PCR) that selectively amplifies a segment of non-metazoan Small Subunit (SSU) rDNA gene was validated. The primers used were: 18S-EUK581-F (5′-GTGCCAGCAGCCGCG-3′) and 18S-EUK1134-R (5′-TTTAAGTTTCAGCCTTGCG-3′) with specificity provided by the 19-base reverse primer. Its target site is highly conserved across the Archaea, Bacteria, and eukaryotes (including fungi), but not most Metazoa (except Porifera, Ctenophora, and Myxozoa) which have mismatches at bases 14 and 19 resulting in poor or failed amplification. During validation, UNonMet-PCR amplified SSU rDNA gene fragments from all assayed protists (n = 16 from 7 higher taxa, including two species of marine phytoplankton) and Fungi (n = 3) but amplified very poorly or not at all most assayed Metazoa (n = 13 from 8 higher taxa). When a non-metazoan parasite was present in a metazoan host, the parasite DNA was preferentially amplified. For example, DNA from the parasite Trypanosoma danilewskyi was preferentially amplified in mixtures containing up to 1,000× more goldfish Carassius auratus (host) DNA. Also, the weak amplification of uninfected host (Chionoecetes tanneri) SSU rDNA did not occur in the presence of a natural infection with a parasite (Hematodinium sp.). Only Hematodinium sp. SSU rDNA was amplified in samples from infected C. tanneri. This UNonMet-PCR is a powerful tool for amplifying SSU rDNA from non-metazoan pathogens or symbionts that have not been isolated from metazoan hosts.

Most previously reported generation times for rumen ciliate protozoa are longer than would be required to prevent their being flushed out of the rumen. In an earlier study from this lab, using a sequential transfer procedure, generation times between 12 and 13 h were determined for both Epidinium caudatum and Entodinium caudatum. This would permit these species to be maintained in a rumen with a fluid volume turnover rate as rapid as twice a day. In this study, generation times were estimated for Entodinium exiguum (13.2 h), Eudiplodinium maggii (26.8 h), and Ophryoscolex purkynjei (29 h), by sequential transfer at both 12 and 24 h time periods. The generation time for E. exiguum is lower than reported for this and other Entodinium species as determined by logarithmic growth from a small inoculum, but similar to that obtained for Ent. caudatum using sequential transfer. Eudiplodinium maggii and O. purkynjei generation times are similar to previous estimates of 24- and 24–48 h, respectively. However, it was observed that after an adaptation period of 36 to 48 h (generally 3–4 transfers) cell concentrations decreased and generation times were markedly decreased, i.e. 12.2 h for Ent. exiguum, 15.0 h for E. maggii and 12.8 h for O. purkynjei. In a separate study, varying both the concentration of Epidinium and the quantity of substrate fed per cell had no effect on generation time.

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 °C and 37 °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 °C than when cultured at 37 °C. However, spores from the insect isolates cultivated at 30 °C or 37 °C infected significantly fewer mammalian cells at 37 °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 °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 °C.

Steady state levels of the HSP70 transcript were followed by Northern hybridization in Moneuplotes crassus in order to investigate the mechanisms of the short term and long term response to heat shock in a spirotrichous ciliate. The influence of inhibitors of transcription or translation on the transcript levels was also studied. The heat shock response could be dissected into two phases. An initial protein-dependent stabilization of the mRNA was followed by an increase of the steady state transcript level that was dependent on continued transcription. As expected, the half-life of the RNA was short. Western blot analysis then showed that the HSP70 protein accumulated only upon permanent heat shock. It is concluded that the regulation of the heat shock response is a two-step process that occurs at the transcript level.

A hypodiploid strain of Tetrahymena thermophila has been obtained that shows arrest at the stage of condensed nuclei, corresponding to metaphase I of normal conjugants and induced arrest at meiotic metaphase I (i.e. at the stage of condensed, bivalent chromosomes) in its wt partner mate. The metaphase I arrested conjugants retained their old macronuclei and most of them underwent cell fusion, instead of separation of exconjugants. The doublets were viable and cortically integrated. When the arrest inducing strain was crossed to the haploid tester strain, the haploid micronuclei were arrested in the meiotic metaphase I as the diploid ones had been; the monovalent, chromosomes were condensed, the arms of sister chromatids were not separated, and they were not segregated. Separation of the arms of sister chromatids and disjunction of bivalent chromosomes were not prerequisite for the formation of microtubular spindles in those cells that were arrested in meiotic metaphase I. After re–feeding, the doublet cells resumed cell divisions, segregating two macronuclei and micronuclei at random. One macronucleus was derived from the arrest inducing strain and the other from the tester strain. Heterokaryon strains with macronuclei derived from the parental arrest inducing strain and with the micronucleus derived from the parental wt tester strain were obtained. Surprisingly, these heterokaryons did not induce meiotic arrest. Thus, the arrest in the meiotic metaphase I was induced by the micronucleus and not by the macronucleus of the arrest inducing strain.

The 82–90 kD family of molecular chaperone proteins has homologs in eukaryotes (Hsp90) and many eubacteria (HtpG) but not in Archaebacteria. We used representatives of all four different eukaryotic paralogs (cytosolic, endoplasmic reticulum (ER), chloroplast, mitochondrial) together with numerous eubacterial HtpG proteins for phylogenetic analyses to investigate their evolutionary origins. Our trees confirm that none of the organellar Hsp90s derives from the endosymbionts of early eukaryotes. Contrary to previous suggestions of distant origins through lateral gene transfer (LGT) all eukaryote Hsp90s are related to Gram-positive eubacterial HtpG proteins. The nucleocytosolic, ER and chloroplast Hsp90 paralogs are clearly mutually related. The origin of mitochondrial Hsp90 is more obscure, as these sequences are deeply nested within eubacteria. Our trees also reveal a deep split within eubacteria into a group of mainly long-branching sequences (including the eukaryote mitochondrial Hsp90s) and another group comprising exclusively short-branching HtpG proteins, from which the cytosolic/ER versions probably arose. Both versions are present in several eubacterial phyla, suggesting gene duplication very early in eubacterial evolution and multiple independent losses thereafter. We identified one probable case of LGT within eubacteria. However, multiple losses can simply explain the evolutionary pattern of the eubacterial HtpG paralogs and predominate over LGT. We suggest that the actinobacterial ancestor of eukaryotes harbored genes for both eubacterial HtpG paralogs, as the actinobacterium Streptomyces coelicolor still does; one could have given rise to the mitochondrial Hsp90 and the other, following another duplication event in the ancestral eukaryote, to the cytosolic and ER Hsp90 homologs.