ALBERT

Notes: The dynein ATPases are a family of motor enzymes that drive microtubule sliding in cilia and flagella and contribute to microtubule-based transport inside cells. the multi-dynein hypothesis makes two predictions: 1) Axonemes contain multiple dynein heavy chain (DHC) isoforms, each encoded by a different gene; 2) Each isoform performs a specific role in ciliary beating. We used PCR-based techniques to clone thirteen different DHC sequences from Tetrahymena genomic DNA. All thirteen genes appeared to be expressed in growing cells. Comparisons of the deduced amino acid sequences of the thirteen DHCs with other known DHCs suggested that we have cloned three outer arm DHCs. two cytoplasmic DHCs, and eight inner arm DHCs.

Notes: Reticulomyxa filosa is a freshwater protist possessing fine granular, branching and anastomosing pseudopodia and therefore traditionally placed in the class Granuloreticulosea, order Athalamida, as a sister group to the order Foraminiferida. Recent studies have revealed remarkable similarities in pseudopodial motility and ultrastructure between R. filosa and foraminifera (e.g. Allogromia laticollaris), prompting us to conduct a molecular phylogenetic analysis of these seemingly disparate organisms. We sequenced the complete small-subunit of the ribosomal DNA of the cultured strain of R. filosa and compared it to the corresponding sequences of other protists including 12 species of foraminifera. We also sequenced and analyzed the actin coding genes from R. filosa and two species of foraminifera, Allogromia sp. and Ammonia sp. the analysis of both data sets clearly shows that R. filosa branches within the clade of foraminifera, suggesting that R. filosa is in fact a freshwater naked foraminiferan.

Notes: We examined the nuclear behavior of mating Tetrahymena cells that had been mechanically disrupted at various times throughout conjugation. Disruption was achieved by agitating conjugating Tetrahymena in the presence of 0.1-3 mm glass beads. Two minutes of agitation with 1 mm beads yielded optimal pair disruption (70%) with high viability (92%). Disrupting pairs between 0-4.7 h after the initiation of mating produced mostly disrupted conjugants in which development was aborted. However, as many as 20% of these early disrupted conjugants completed development even without their mating partners. After 5 h the percentage of disrupted conjugants completing development increased dramatically, reaching 80% by 6.7 h. These results support a model suggesting that events associated with nuclear exchange and fusion 5 h into conjugation trigger a commitment to completion of the postzygotic developmental program. the early conjugants that completed development following disruption suggest that development can be sustained even in the absence of a mating partner. This represents a novel method of bringing the micronuclear genome into macronuclear expression with minimal cytoplasmic exchange between partners. We discuss these results in light of a model relating cortical and nuclear signaling events that reciprocally drive conjugal development.

Notes: 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.

Notes: We have characterized a macronuclear gene of the ciliate protozoan Euplotes raikovi, which encodes an acidic ribosomal protein of the P protein family. This gene shows the typical organization of the hypotrich ciliate macronuclear “gene-sized” molecules with Euplotes telomeres at the ends. the longest open reading frame encodes a conceptual protein of 113 amino acid residues, with a molecular mass and pI value of 11.45 kDa and 3.97, respectively. By using sequence homology analysis, the protein was found to belong to the ribosomal P2 protein family and was named Er P2, where Er stands for Euplotes raikovi. These proteins, generally called A (acidic/alanine rich) proteins in prokaryotes and P (phosphorylated) proteins in eukaryotes, in which they are divided into P1 and P2 families, play a role in the elongation step of protein synthesis. Approximately 40% amino acid sequence identity was found between the cloned protein and other known protozoan ribosomal P2 proteins. Within its N-terminal half, this protein contains several potential kinase phosphorylation sites. Protein Er P2 differs markedly from the consensus P protein sequence in its C-terminal region, usually highly conserved among eukaryotic ribosomal P proteins, and shows similarities with the C-terminus of the archaebacterial ribosomal A proteins. to our knowledge, this E. raikovi protein represents the first demonstration of a ribosome-associated protein of the P2 family in a ciliate protozoan.

Notes: The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis. peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. the amino acid composition of Chlamydomonas lysinerich histones was compared to those of various other unicellular algae.

Notes: Sultanophrys arabica and Tracheloraphis sp., two interstitial karyorelictid ciliates, were cultivated in sealed 100–200 ml glass bottles half-filled with filtered interstitial water to which some millilitres of the natural organism community and a couple of wheat grains were added. Removing sand grains and sealing the bottles were crucial to achieve a low oxygen tension milieu, which was maintained by the algae contained in the community. This cultivation method provided, for the first time, rich cultures with many feeding, dividing, and conjugating cells. Both species were omnivorous and fed through the apical end, where a well-developed oral apparatus is present. Apical feeding was documented by micrographs of living specimens and by scanning electron microscopy of preserved cells.

Notes: Microsporidia form a large and ubiquitous group of obligately intracellular parasitic eukaryotes, increasingly recognized as pathogens in humans. Transmission of invertebrate microsporidia to mammals has been considered impossible because temperature seemed to be a limiting factor for development. Nosema algerae, a microsporidian of anopheline mosquitoes, was cultured in human muscle fibroblasts at temperatures of 31° C and 38° C. This is the first record of an invertebrate microsporidian developing in human cells at a temperature above 36° C. The ultrastructure of N. algerue growing in human muscle fibroblasts is similar to that of Bruchiola vesicularum, a microsporidian species previously described in the muscle of an AIDS patient.

Notes: Fish trypanosomes have traditionally been classified according to the host species from which they were isolated, each isolate being regarded as a distinct species. To test the soundness of this practice, the genetic variabilities of the kinetoplast 12S rRNA-encoding genes of different fish trypanosomes isolates were compared. The DNAs were extracted from trypanosomes cloned from blood samples of 15 donors representing ten different fish species in four orders from waters of three major river systems of Central and Northern Europe. Comparison with other trypanosomatid sequences revealed that the fish trypanosomes form a monophyletic group with Trypanosoma brucei as a sister group. Pairwise comparisons of genetic distances yielded a wide range of continuous variation with no indication of any discontinuities attributable to barriers to gene flow. The genetic distances did not correlate with either the identity of the host species or geography. The host specificity of fish trypanosomes appears to be limited.

Notes: A novel microsporidian parasite is described, which infects the crustacean host Gammarus duebeni. The parasite was transovarially transmitted and feminised host offspring. The life cycle was monomorphic with three stages. Meronts were found in host embryos, juveniles, and in the gonadal tissue of adults. Sporoblasts and spores were restricted to the gonad. Sporogony was disporoblastic giving rise to paired sporoblasts, which then differentiated to form spores. Spores were not found in regular groupings and there was no interfacial envelope. Spores were approximately 3.78 × 1.22 μm and had a thin exospore wall, a short polar filament, and an unusual granular polaroplast. All life cycle stages were diplokaryotic. A region from the parasite small subunit ribosomal RNA gene was amplified and sequenced. Phylogenetic analysis based on these data places the parasite within the genus Nosema. We have named the species Nosema granulosis based on the structure of the polaroplast.

Notes: Interaction between factors from Trypanosoma cruzi extracts and AP-1 sequences was studied by electrophoretic mobility shift assays. Using a double-stranded probe carrying the AP-1 sequence from the SV40 promoter, three specific complexes designated A, B, and C were detected. Complexes A and C were formed when using single-stranded probes. The relative amount of complex B, specific for double-stranded DNA, increased as a function of probe length. Complexes were stabilized by cross-linking with UVC irradiation and resolved on denaturing SDS-PAGE. Complex A generated bands of 60- and 39 kDa; complex B produced two bands of 46- and 43 kDa; and complex C generated one band of 43 kDa. The AP-I binding activity was much higher in purified nuclear preparations than in soluble fractions, and was detected in crude extracts from the three forms of the parasite. The binding signal, however, was much stronger in amastigote and trypomastigote than in the epimastigote forms. Specific binding was increased by oxidative stress. Antibodies raised against peptides corresponding to conserved domains of mammalian c-Jun and c-Fos detected bands of 40- and 60 kDa, respectively, in a nuclear epimastigote preparation.

Notes: Malawimonas jakobiformis n. gen., n. sp., is established for a bacterivorous heterotrophic nanoflagellate isolated from the Malawi shore of Lake Nyasa (eastern Africa). Trophic stages observed were anteriorly biflagellate and naked. The posterior flagellum of a trophic cell resided in a conspicuous groove on the ventral surface, and bore a prominent vane. A Golgi stack and a mitochondrion with discoidal cristae were present anterior to the nucleus. The kinetid consisted of two short, slightly separated basal bodies, four microtubular roots, and associated fibers and bands. The three microtubular roots associated with the posterior basal body were associated with the ventral groove, while the single root associated with the anterior basal body gave rise to secondary cytoskeletal microtubules. Dividing cells became rounded, with persistent flagella. Cysts were uninucleate, and had thin organic walls without clearly differentiated apertures or ornamentation but with conspicuous attachment pads. Kinetid elements were present within cysts. On the basis of microscopical features, especially those of the kinetid, the nearest relatives of M. jakobiformis are the mitochondriate “jakobid” protists (families Histionidae and Jakobidae) and the amitochondriate retortamonads. Malawimonadidae n. fam. is established to accommodate this species.

Notes: In order to investigate the physiological potentialities of behaviorally inert Oxytricha bifaria, cooled from 24 to 9° C according to an already standardized protocol, a warm microgradient was created in the experimental chamber and the behavior of ciliates was analyzed both at the level of the passing warm wave front (dynamic microgradient), and, afterwards, when the thermal gradient stabilized (static microgradient). We monitored the general behavior of the experimental populations by means of (i) their centroid, (ii) the ethograms of single oxytrichas, and (iii) calculating the numerical indices and rates of their creeping tracks. It was found that (a) the population moves towards the heating source, (b) the oxytrichas react immediately to the thermal stimulus, (c) creeping forwards (d) at very high velocity (e) along uninterrupted looping tracks (f) according to precise mechanisms of positive/negative orthokinesis, thus orientating towards the environmental optimum. Moreover, (g) the ciliates accumulate in the warmest area, correcting their creeping by means of many specific behavioral patterns (the Side Stepping Reaction) once the gradient is stabilized. At 9° C, despite their inertness, the ciliates are still able to behave adaptively reacting immediately and orientatedly, once a directional factor (the thermal gradient) arises in an isotropic environment.

Notes: Sequencing of the Trypanosoma cruzi genome is underway. Expressed sequence tags, obtained from cDNA libraries, facilitate mapping and gene discovery. The efficiency of large-scale generation of such tags is increased when using normalized cDNA libraries, where the frequency of individual clones is brought within a narrow range. Repetitive sequencing of abundant clones is therefore minimized. We constructed a normalized cDNA library from epimastigotes of clone CL Brener, and the efficiency of normalization of representative clones was assessed and shown to be adequate. The normalized cDNA library has been distributed to several groups and large-scale sequencing is currently in progress.

Notes: Utraviolet radiation has provided an evolutionary challenge to life on Earth. Recent increases in surficial ultraviolet B fluxes have focused attention on the role of UV radiation in protistan ecology, cancer, and DNA damage. Exploiting this new wealth of data, I examine the possibility that ultraviolet radiation may have played a significant role in the evolution of the first eukaryotes, that is, protists. Protists probably arose well before the formation of a significant ozone shield, and thus were probably subjected to substantial ultraviolet A, ultraviolet B, and ultraviolet C fluxes early in their evolution. Evolution consists of the generation of heritable variations and the subsequent selection of these variants. Ultraviolet radiation has played a role both as a mutagen and as a selective agent. In its role as a mutagen, it may have been crucial in the origin of sex and as a driver for molecular evolution. As a selective agent, its influence has been broad. Discussed in this paper are the influence of ultraviolet radiation on biogeography, photosynthesis, and desiccation resistance.

Notes: The Organelle Genome Megasequencing Program (OGMP) investigates mitochondrial genome diversity and evolution by systematically determining the complete mitochondrial DNA (mtDNA) sequences of a phylogenetically broad selection of protists. The mtDNAs of lower fungi and choanoflagellates are being analyzed by the Fungal Mitochondrial Genome Project (FMGP), a sister project to the OGMP. Some of the most interesting protists include the jakobid flagellates Reclinomonas americana, Malawimonas jakobiformis, and Jakoba libera, which share ultrastructural similarities with amitochondriate retortamonads, and harbor mitochondrial genes not seen before in mtDNAs of other organisms. In R. americana and J. libera, gene clusters are found that resemble, to an unprecedented degree, the contiguous ribosomal protein operons str, S10, spc, and alpha of eubacteria. In addition, their mtDNAs code for an RNase P RNA that displays all the elements of a bacterial minimum consensus structure. This structure has been instrumental in detecting the rnpB gene in additional protists. Gene repertoire and gene order comparisons as well as multiple-gene phylogenies support the view of a single endosymbiotic origin of mitochondria, whose closest extant relatives are Rickettsia-type α-Proteobacteria.

Notes: The biggest unsolved problems in chloroplast evolution are the origins of dinoflagellate and euglenoid chloroplasts, which have envelopes of three membranes not two like plants and chromists, and of the sporozoan plastid, bounded by four smooth membranes. I review evidence that all three of these protozoan plastid types originated by secondary symbiogenesis from eukaryotic endosymbionts. Instead of separate symbiogenetic events, I argue that dinoflagellate and sporozoan plastids are directly related and that the common ancestor of dinoflagellates and Sporozoa was photosynthetic. I suggest that the last common ancestor of all Alveolata was photosynthetic and acquired its chlorophyll c-containing plastids in the same endosymbiogenetic event as those of Chromista. Chromista and Alveolata are postulated to be a clade designated chromalveolates. I propose that euglenoids obtained their plastids from the same (possibly ulvophycean) green alga as chlorarachneans and that Discicristata (Euglenozoa plus Percolozoa) and Cercozoa (the group including chlorarachneans) form a clade designated cabozoa (protozoa with chlorophyll a + b). If both theories are correct, there were only two secondary symbiogenetic events (witnessed by the chlorarachnean and cryptomonad nucleomorphs) in the history of life, not seven as commonly assumed. This greatly reduces the postulated number of independent origins of chloroplast protein-targeting machinery and of gene transfers from endosymbiont to host nuclei. I discuss the membrane and plastid losses and innovations in protein targeting implied by these theories, the comparative evidence for them, and their implications for eukaryote megaphylogeny. The principle of evolutionary conservatism leads to a novel theory for the function of periplastid vesicles in membrane biogenesis of chlorarachneans and chromists and of the key steps in secondary symbiogenesis. Protozoan classification is also slightly revised by abandoning the probably polyphyletic infrakingdom Actinopoda, grouping Foraminifera and Radiolaria as a new infrakingdom Retaria, placing Heliozoa within a revised infrakingdom Sarcomastigota, establishing a new flagellate phylum Loukozoa for Jakobea plus Anaeromonadea within an emended subkingdom Eozoa, and ranking Archezoa as an infrakingdom within Eozoa.

Notes: Trichomonads, together with diplomonads and microsporidia, emerge at the base of the eukaryotic tree, on the basis of the small subunit rRNA phylogeny. However, phylogenies based on protein sequences such as tubulin are markedly different with these protists emerging much later. We have investigated 70 kDa heat-shock protein (HSP70), which could be a reliable phylogenetic marker. In eukaryotes, HSP70s are found in cytosol, endoplasmic reticulum, and organelles (mitochondria and chloroplasts). In Trichomonas vaginalis we identified nine different HSP70-encoding genes and sequenced three nearly complete cDNAs corresponding to cytosolic, endoplasmic reticulum, and mitochondrial-type HSP70. Phylogenies of eukaryotes were reconstructed using the classical methods while varying the number of species and characters considered. Almost all the undoubtedly monophyletic groups, defined by ultrastructural characters, were recovered. However, due to the long branch attraction phenomenon, the evolutionary rates were the main factor determining the position of species, even with the use of a close outgroup, which is an important advantage of HSP70 with respect to many other markers. Numerous variable sites are peculiar to Trichomonas and probably generated the artefactual placement of this species at the base of the eukaryotes or as the sister group of fast-evolving species. The inter-phyla relationships were not well supported and were sensitive to the reconstruction method, the number of species, and the quantity of information used. This lack of resolution could be explained by the very rapid diversification of eukaryotes. likely after the mitochondrial endosymbiosis.

Notes: Pseudocohnilembus species exhibit a polymorphic life cycle consisting of trophic cells, theronts, and cysts. Pseudocohnilembus pusillus isolated from the intertidal mats of Laguna Figueroa, Baja California Norte, Mexico, forms desiccation-resistant cysts in response to bacterial food depletion. This isolate is a euryhaline organism, able to grow at salinities from freshwater to 96 ppt total salinity and from pH 6–9. Electron micrographs show that oral and somatic cilia and kinetids are retained inside young cysts. Cyst walls are composed of a single layer (0.1 μm) of granular material. Under all conditions, as bacterial food was depleted, P. pusillus cells formed cysts, except for a small proportion (1–5%) that continued to swim. Changes in pH and salinity did not directly induce cyst formation. Salinity did greatly affect growth rate. Doubling times were shortest at 16 ppt salinity and at pH 7–8. Cyst formation occurred later in the growth cycle as more food bacteria were added. Additionally, ciliates grown in small culture volumes (10 ml) formed cysts sooner than cultures in larger volumes (100 ml), suggesting that crowding may influence cyst formation. Mature cysts may survive desiccation at least as long as one month at 37° C and for as long as one year at 20 ± 3° C. Although trophic cells did not survive desiccation or anoxia, encysted ciliates from liquid stationary phase cultures kept in anoxic seawater for one month excysted into swimming cells within 2.5 h after exposure to air. The adaptability of P. pusillus to extremes of salinity, pH, desiccation, and anoxia permits survival in its environmentally variable, microbial mat habitat.

Notes: Two dyneins can be extracted from Tetrahymena ciliary axonemes. The 22S dynein contains three heavy chains (HC), sediments at 22S in a sucrose gradient, and makes up the outer arms. The 14S dynein contains two to six HCs, sediments at 14S, and is thought to contribute to formation of the inner arms. We have identified two large proteins that are extracted from Tetrahymena axonemes with high salt and that sediment together at approximately 18S. The two large proteins cleave when subjected to UV light in the presence of ATP and vanadate, suggesting both proteins are dynein HC. Antibodies against one of the 18S HCs do not recognize 22S dynein HCs. Antibodies to 22S dynein HC do not bind appreciably to 18S dynein photocleavage fragments. Taken together, these results indicate that the large proteins that sediment at 18S are axonemal dynein heavy chains.

Notes: Aerobic and anaerobic ciliates swim towards the cathode when they are exposed to a constant DC field. Nyctotherus ovalis from the intestinal tract of cockroaches exhibits a different galvanotactic response: at low strength of the DC field the ciliates orient towards the anode whereas DC fields above 2–4 V/cm cause cathodic swimming. This reversal of the galvanotactic response is not due to backward swimming. Rather the ciliates turn around and orient to the cathode with their anterior pole. Exposure to various cations, chelators, and Ca2--channel inhibitors suggests that Ca2--channels similar to the “long lasting” Ca2--channels of vertebrates are involved in the voltage-dependent anodic galvanotaxis. Evidence is presented that host-dependent epigenetic factors can influence the voltage-threshold for the switch from anodic to cathodic swimming.

Notes: Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morphologically different Perkinsus species isolates from the gill (G117) and the hemolymph (H49) of the softshell clam, Mya arenaria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G117 and H49 indicated that these genes are 1803 and 1806 base-pair long, respectively. A sequence similarity of 〉 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes-Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor-Joining method, we showed that G117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.

Notes: The internally transcribed spacer regions 1 and 2 (ITS-1 and ITS-2) and the 5.8S ribosomal RNA gene of Isotricha prostoma were examined for intraspecific sequence variation. There were no differences in the ITS-1/5.8S/ITS-2 region among cattle and sheep isolates of I. prostoma from Australia, Canada, and the United States, indicating that this region is 100% conserved among eight isolates from two continents.

Notes: . To survive and replicate in vertebrate hosts, protozoan and fungal invaders must be capable of securing host iron. Successful pathogens obtain the metal from either extraction of heme, binding of siderophilins. binding of siderophores, and/or iron pools within host cells. the actual strategy can vary with the availability of iron in the particular host milieu. As a corollary, hosts have developed an elaborate iron withholding defense system. Conditions that can compromise the system as well as procedures that can strengthen it are reviewed.

Notes: Encephalitozoonidae are microsporidia associated with human infections including hepatitis, encephalitis, conjunctivitis, and disseminated disease. Microsporidia produce a small resistant spore containing a polar tube which serves as a unique vehicle of infection. Polar tube proteins (PTPs) from Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, and Encephalitozoon cuniculi were purified to homogeneity by HPLC. By SDS-PAGE, the Mr of E. hellem PTP was 55 kDa, while the Mr of E. intestinalis and E. cuniculi PTP was 45 kDa. Polyclonal rabbit antiserum to these purified PTPs localized to polar filaments by immunogold electron microscopy and immunofluorescence, and demonstrated cross-reactivity by both immunoblotting and immunogold electron microscopy. These PTPs have similar solubility properties, hydrophobicity, and proline content to a 43-kDa PTP we have previously purified from Glugea americanus, a fish microsporidium. As the polar tube is critical in the transmission of this organism, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.

Notes: We have dissected the topoisomerase II gene of members of the two recently characterized subgroups of Trypanosoma cruzi to obtain further evidence to support this dichotomy of isolates in this important parasite. Pulsed field gel electrophoresis showed a striking heterogeneity in the molecular karyotypes of the strains analyzed. Southern analysis of these chromosome gels also showed heterogeneity in the size and number of chromosomes containing the topoisomerase II gene. Analysis of DNA restriction fragment length polymorphisms of the topoisomerase II gene also showed two principal patterns consistent with the two previously characterized groups. Finally, the sequences of portions of the topoisomerase II genes from members of the T. cruzi groups showed two distinct patterns, again consistent with the previous grouping of this parasite. Thus, this work clearly supports previous observations suggesting an ancient divergence of known T. cruzi isolates into two main branches.

Notes: Sodium orthovanadate at 0.1–5.0 mM affected cell proliferation of Tetrahymena in a dose-dependent manner. At 1 h the cell increment was 76–12% of the control (100%), but after lag periods in 1–5 mM the growth rate remained at 76% of control in 0.1 mM vanadate and at 64–61% of control in 0.2–5.0 mM vanadate. Endocytosis was affected in both a time- and dose-dependent manner; an increasing number of cells did not form vacuoles. Cell motility increased initially in 0.1 mM vanadate but decreased later as it did in 0.5–2.0 mM vanadate where the proportion of immobile cells increased with time. Cell divisions occurred at all concentrations but macronuclear elongation was disturbed and subsequent cytokinesis resulted in daughter cells containing the entire G2 macronucleus, a large or small portion of it, or no nucleus at all. Moreover, odd cell shapes appeared with time. The size of the cell and nucleus increased but there was great variation with disturbed cytoplasm/nucleus ratios. Treated cells had dilated rough endoplasmic reticulum that included dense material, presumed to be vanadate. which was not seen in control cells. Scant amounts of dense material were found in dense granules, small vacuoles, and abundantly in contractile vacuoles. It is argued that interference with proper microtubular function is the main effect of vanadate.

Notes: During conjugation of Paramecium caudatum, nuclear determination occurs soon after the third postzygotic division: one of the four anterior nuclei becomes the micronucleus and the remaining three degenerate, while four posterior nuclei differentiate into macronuclear anlagen. Macronuclear differentiation is supposed to be dependent on a cytoplasmic differentiation factor. In this study, postzygotic cells were subjected to heat shock for 30 min and nuclear changes were observed by staining with carbol fuchsin solution. When heat shock was initiated during the period from metaphase to telophase of the third postzygotic division, cells showed an excess of macronuclear anlagen and were typically amicronucleate. Abnormal nuclear localization around the end of the third (last) postzygotic division may explain the origin of these kinds of cells. A similar phenomenon appeared after treatment with actinomycin D or emetine. Since heat shock did not inhibit macronuclear differentiation but destroyed the formation of micronuclei, some factor(s) probably plays an essential role in nuclear determination, especially in the protection of the micronuclei.

Notes: Methods for mass transformation of Paramecium tetraurelia were established using plasmids bearing neomycin-resistance or calmodulin gene fragments. Phenotypic and molecular analyses showed that, although variable, up to 5% transformation can be achieved by electroporation. Concentrations of divalent cations Ca2- and Mg2+ in the electroporation medium were crucial for efficient transformation. Strong neomycin-resistance transformation using bioballistic particle bombardment with gold particles was observed. For both methods, hybridization to transformant DNA revealed plasmid signals consistent with macronuclear transformation and correlated with transformed phenotypes. Complementation of a known calmodulin gene mutation was also achieved by mass transformation. Possible sources of variation and the general utility of these methods are discussed.

Notes: We investigated growth and grazing rates of Strombidinopsis sp. when feeding on several species of red-tide and/or toxic dinoflagellates. Strombidinopsis sp. one of the largest aloricate choreotrichs so far reported, grew well on Lingulodinium polyedrum, Gymnodinium sanguineum, Scrippsiella trochoidea, Cochlodinium polykrikoides, and Prorocentrum minimum, but failed to grow on Amphidinium carterae. Specific growth rates of Strombidinopsis sp. increased rapidly with increasing prey density up to ca. 100 ng C ml-1, but were saturated or increased slightly at higher concentrations. Maximum specific growth rates of Strombidinopsis sp. on various prey species were 1.38 day-1 for C. polykrikoides, 1.27 for G. sanguineum, 1.06 for P. minimum, 0.83 for L. polyedrum, and 0.67 for S. trochoidea. Threshold prey concentrations (where net growth = 0) were 12–38 ng C ml-1. Maximum ingestion and clearance rates of Strombidinopsis sp. were 353 ng C grazer-1 day-1 and 110 μ, l grazer-1 h-1, respectively. Strombidinopsis sp. exhibited higher maximum growth, ingestion, and clearance rates than the mixotrophic dinoflagellate Fragilidium cf. mexicanum or the heterotrophic dinoflagellates Protoperidinium cf. divergens and P. crassipes, when grown on the same prey species. In addition, the sequence of prey species arranged according to growth response of Strombidinopsis sp. differed considerably from those of Fragilidium cf. mexicanum, Protoperidinium cf. divergens, and P. crassipes.

Notes: We have previously shown that the cell death of Tetrahymena thermophila in low inocula cultures in a chemically-defined medium is not apoptotic. The death is caused by a cell lysis occurring at the medium-air interface and can be prevented by the addition of insulin or Pluronic F-68. Here, we report that cell death can also be caused by the medium. The specific effects of several medium constituents were tested in the presence and absence of an interface. Four of the 19 amino acids (arginine, aspartic acid, glutamic acid, and histidine in millimolar concentration) as well as Ca2+ (68 μM) and Mg2+ (2 mM) and trace metal ions (micromolar concentrations) are all sufficient to induce the interface-mediated death. The effect of the amino acids and the salt ions Ca2+ and Mg2+ can be abolished by the addition of insulin (10-6 M) or Pluronic F-68 (0.01% w/v), whereas insulin/Pluronic F-68 only postpones the death induced by trace metal ions. On the basis of our findings, a new recipe for a chemically-defined medium has been formulated. Single cells can grow in this medium in the presence of medium-air interface without any supplements.

Notes: We have devised a two step method for the synchronous induction of telotrochs in the peritrich ciliate, Vorticella convallaria. The method is easy, reliable, and allows us to study the earliest events of telotroch formation at the ultrastructural, biochemical, and molecular levels. The steps involved are: (1) excising the cell body from the stalk in a large population (7.4 times 104 cells) of EDTA-treated, attached cells by the application of monocalcium phosphate monohydrate solution at pH 3.2, (2) rinsing and suspending the isolated cell bodies in inorganic medium. Within 90 min, 80% of the population forms telotrochs. Analysis of factors that are important for maximum stalk excision and transformation shows that the population must not be older than 2 d and the most effective concentration of monocalcium phosphate is 4.8 mM for a 20 min exposure. The most effective monocalcium phosphate is in the monohydrated form. A pH value of 3.2, produced by the addition of hydrochloric acid in the presence or absence of calcium is not sufficient to initiate stalk excision and telotroch formation. This observation leads us to conclude that stalk excision is dependent on monocalcium phosphate or its hydrolysis products.

Notes: The morphology and infraciliature of two new, mycophagous soil ciliates are described. Specimens were isolated from dried, rewetted soil samples with the non-flooded Petri dish method and investigated in vivo and with various silver impregnation techniques. Fungiphrya strobli n. g., n. sp. was discovered in the mud of rock-pools on the summit of Table Mountain, Republic of South Africa. It is a holotrichously ciliated, about 50 × 40 μm-sized grossglockneriid with the oral apparatus on the right side of the cell. The adoral ciliature, minute in all other members of the group, is well developed and has a mean of eight kineties forming a conspicuous left, oral polykinetid, highly reminiscent of that found in small species of the genus Colpoda. The ejected extrusomes have a unique, inflated distal end. Grossglockneria ovata n. sp. was discovered in leaf litter from the Lackawanna State Forest in Pennsylvania, USA. It differs from the other members of the genus by the ovate shape, smooth cortex, and the sparse, irregularly-shaped mucocysts. Taxonomic characters and ranking of grossglockneriids are discussed. Because of the complex, unique feeding tube associated with a unique feeding strategy, mycophagy, it is argued that grossglockneriid ciliates should be classified in a separate order, in spite of their close genetic relatedness to members of the order Colpodida.

Notes: High metacyclogenesis was induced when freshly-isolated Trypanosoma rangeli from humans were grown in a modified liver-infusion-tryptose medium and transferred into the medium overlaid on mouse fibroblasts at 27° C in a 5% CO2 atmosphere. Such in yitro-generated metacyclic trypomastigotes could induce a significantly high and constant parasitemia in both ICR and SCID mice for a period of about a week but thereafter the parasitemia gradually decreased. Histological examination could not detect any tissue-forms of T. rangeli in various organs of SCID mice. On the other hand, two long-maintained stocks of T. rangeli produced lower metacyclogenesis and only latent parasitemia in both strains of mice. When these populations were incubated in fibroblast cultures at 37° C in a 5% CO2 atmosphere, only trypomastigotes survived for two to three weeks without proliferation, while other forms, mainly epimastigotes, soon began to swell and degenerate. Electron microscopy showed that most surviving trypomastigotes had the basket-like conformation of the kinetoplasts. This is characteristic of the non-dividing trypomastigote stage of T. cruzi, and suggests that T. rangeli trypomastigotes may survive long periods in the blood without proliferation.

Notes: Several transmission studies, as well as recent molecular data, have indicated that the two classes Myxosporea and Actinosporea represent different life cycle stages of Myxozoa. To evaluate the life cycles of myxozoa in catfish aquaculture systems, the small subunit (18S) ribosomal RNA gene sequences of Henneguya exilis, a myxosporean from channel catfish Ictalurus punctatus, and an actinosporean (previously designated as Aurantiactinomyxon janiszewskai) from the aquatic oligochaete Dero digitata were determined. The sequences were identical, indicating that H. exilis and the actinosporean are alternate life stages of a single species. This is the first report identifying the actinosporean stage of the genus Henneguya.

Notes: The mechanism of stomatin-induced differentiation of Tetrahymena vorax was investigated by in vivo protease degradation of cell surface proteins, the direct measurement of products formed from the activation of phospholipase C, and the use of an array of signal transduction inhibitors/activators. The data indicate that a surface-exposed protein is required for stomatin to signal the cells to differentiate and that the cells are committed to the differentiation pathway within two hours after exposure to stomatin. Analysis of radiolabeled polyphosphoinositols and inositol lipids from control and stomatin-treated populations in the presence of 10 mM LiCl were consistent with a rapid activation of phospholipase C. Within five min following addition of stomatin, this resulted in an increase in polyphosphoinositols and a concomitant decrease in the relative amounts of phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate.

Notes: Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82 kDa surface glycoprotein (gp82) that has been implicated in the mammalian cell invasion. When the non-infective epimastigote stage of the parasite was transfected with a vector containing the gp82 gene, an 82 kDa surface glycoprotein, which was indistinguishable from the metacyclic stage protein, was expressed. In contrast, when the same gene was expressed in transfected mammalian cells, although a large amount of protein was produced, it was not imported into the endoplasmic reticulum and glycosylated. This blockage in targeting and processing could be partially compensated for by the addition of a virus haemagglutinin signal peptide to the amino terminus of gp82. Thus, the requirements for membrane protein processing are distinct in mammals and T. cruzi, and an intrinsic feature of the gp82 prevents subsequent sorting to the mammalian cell surface. These results could be useful in the development of new DNA vaccines against T. cruzi employing parasite genes encoding immunodominant surface glycoproteins.

Notes: Detailed data on bacterial and protistan microfossils are presented from a 0.003 mm3 piece of Triassic amber (Schlierseerit, Upper Triassic period, 220-230 million years old). This microcenosis, which actually existed as such within a very small, probably semiaquatic habitat, included the remains of about two bacteria species, four fungi (Palaeodikaryomyces baueri, Pithomyces-like conidia, capillitium-like hyphae, yeast cells) two euglenoids, two chlamydomonas (Chlamydomonas sp., Chloromonas sp.), two coccal green microalgae (Chlorella sp., Choricystis-like cells), one zooflagellate, three testate amoebae (Centropyxis aculeata var. oblonga-like, Cyclopyxis eurystoma-like, Hyalosphenia baueri n. sp.), seven ciliates (Pseudoplaryophrya nana-like, Mykophagophrys terricola-like, Cyrtolophosis mucicola-like, Paracondylostoma sp., Bryometopus triquetrus-like. Tetrahymena rostrata-like, Paramecium triassicum n. sp.) the microfossils correspond to or diverge from extant species only slightly.

Notes: The eukaryotic cell cycle is regulated by the sequential activation of different CDK/cyclin complexes. Two distinct classes of mitotic cyclin homologues, CYC1 and CYC2, have been identified and cloned for the first time in the ciliate Paramecium. Cyc1 is 324 amino acids long with a predicted molecular mass of 38 kDa, whereas Cyc2 is 336 amino acids long with a predicted molecular mass of 40 kDa. They display 42-51% sequence identity to other eukaryotic mitotic cyclins within the ‘cyclin box’ region. the conserved ‘cyclin box’ and ‘destruction box’ elements can be identified within each of the sequences. Genomic Southern blot analysis indicated that the CYC1 gene has two isoforms, with 92.3% and 85.9% identity at the amino acid level and at the necleotide level, respectively. Both Cyc1 and Cyc2 proteins showed characteristic patterns of accumulation and destruction during the vegetative cell cycle, with Cyc1 peaking at the point of commitment to division (PCD), and Cyc2 reaching the maximal level late in the cell cycle. Immunoprecipitation experiments with antibodies specific to Cyc1 and Cyc2 indicated that Cyc1 and Cyc2 associate with distinct CDK homologues. Both immunoprecipitates exhibited histone H1 kinase activity that oscillated in the cell cycle in parallel with the respective amount of cyclins present. Histone H1 kinase activity associated with Cyc1 reached a peak at PCD while Cyc2 showed maximal activity when about 75% cells have completed cytokinesis. We propose that Cyc1 may be involved in commitment to division, in association with the CDK that binds to p13suc1, Cdk3, and that the Cyc2/Cdk2 complex may regulate cytokinesis. PCR-amplification revealed similar sequences in Tetrahymena, Sterkiella, Colpoda and Blepharisma. suggesting the conservation of the cyclin genes within ciliates. Although cell cycle regulation in ciliates differs in some respects from that of other eukaryotes, the cyclin motifs have clearly been conserved during evolution.

Notes: A very specific L-arginine transporter showing high affinity has been characterized in Trypanosoma cruzi epimastigotes. Uptake was found to be dependent on L-arginine concentration and it was saturable. Values for maximum velocity and Km ranged between 48.1-57.5 pmol·min-1 per 3 times 10- cells and between 4.2-5.5 μM, respectively. the calculated activation energy and Q10 were 31.1 KJ·mol-1, and 1.7, respectively. Uptake velocity significantly increased when cells were preincubated in the absence of L-arginine, Cells retained the labeled amino acid independently of the presence or absence of exogenous L-arginine. the specificity of L-arginine uptake was demonstrated by competition assays in the presence of 80-fold molar excess of natural amino acids and several L-arginine derivatives. the highest levels of inhibition were caused by L-homoarginine, D-arginine, L-canavanine, L-ornithine, and L-citrulline. L-arginine uptake by T. cruzi epimastigotes was not affected by the presence of potassium or sodium ions in the incubation mixture or by pH changes in the range between 5.5-8.5. the major product of L-arginine uptake was characterized as phosphoarginine. Moreover, arginine kinase activity was detected in soluble extracts from T. cruzi epimastigotes.

Notes: Consumption of pine needles tends to cause abortion in domestic cattle but not in elk. the present study was undertaken to determine whether this difference was associated with the rumen microbial population. After emptying the rumen, pregnant cattle were inoculated with either elk or cattle rumen contents. For those cows fed the pine needle diet, there was no difference in abortion rate between those inoculated with rumen contents from either elk or cattle. Protozoal concentrations and number of genera were observed to decrease markedly in all cows fed the diet containing pine needles. the cycloposthiid ciliate Parentodinium africanum was observed in rumen contents from several of the domestic cattle (Bos taurus). Concentrations ranged from 1.4 to 130.6 × 104 per ml of rumen contents, which comprised 4.6 to 80.3% of the total ciliate population. Mean dimensions of this species were: length, 33.4 μm: width, 19.7 μm: length/width ratio, 1.70. which were similar to those previously reported for this species from Bos indicus in Brazil. This is the first observation of P. africanum. originally observed and described in stomach contents of the hippopotamus, either in Bos taurus or in and host in the northern hemisphere.

Notes: Labyrinthulids and thraustochytrids are unicellular heterotrophs, formerly considered as fungi, but presently are recognized as members in the stramenopiles of the kingdom Protista sensu lato. We determined the 18S ribpsomal RNA gene sequences of 14 strains from different species of the six genera and analyzed the molecular phylogenetic relationships. the results conflict with the current classification based on morphology, at the genus and species levels. These organisms are separated, based on signature sequences and unique inserted sequences, into two major groups, which were named the labyrinthulid phylogenetic group and the thraustochytrid phylogenetic group. Although these groupings are in disagreement with many conventional taxonomic characters, they correlated better with the sugar composition of the cell wall. Thus, the currently used taxonomic criteria need serious reconsideration.

Notes: The occurrence of Actinophrys sol, a planktonic heliozoan, in Chesapeake Bay was monitored over a four-year period (1988–1991). Actinophrys sol was widely-distributed throughout Chesapeake Bay and could exceed densities of 5,000 cells liter −1. It was most abundant during the warmer months. Feeding experiments were conducted with field populations of heliozoa using 1-μm fluorescent microspheres to label ciliate prey. Two ciliates, a small Strobilidium sp. (30 μ in diameter) and a Pleuronema sp. (45 μ length), were the primary ciliate-prey items in the water column when the experiments were conducted, although a wide range of ciliate taxa was ingested. Two other ciliates not present in situ, a Cyclidium sp. (20 μ length) and a Uronema sp. (40 μ length), were also labeled and added at various concentrations to field populations of plankton containing A. sol. Heliozoan ingestion rates on in situ prey at concentrations of 30 Strohilidium and one Pleuronema ml−1 were 0.2 to 0.3 prey heliozoan−1 hour−1. Ingestion rates increased to a maximum of 1.2 prey heliozoan −1 hour−1 with additions of 100 Uronema ml−1. A mean clearance rate of 0.15 ml heliozoan−1 day−1 did not change with increasing prey abundance. The abundance and distribution of A. sol suggests that these sarcodines may exert strong grazing pressure on the planktonic ciliate populations of Chesapeake Bay at certain times of the year, and may be important in shaping the ciliate community composition and distribution.

Notes: A macronuclear gene-sized molecule carrying an actin gene from the hypotrich ciliate, Histriculus cavicola, was characterized. Southern blot analysis using a coding region probe suggested that actin in H. cavicola is encoded by a single gene. A comparison of the promoter regions indicated that the H. cavicola actin gene has a TATA box in the 51 flanking region in a position identical to those in other oxytrich ciliates. The coding sequence of this gene is not interrupted by any introns, and codes for a protein of 375 amino acid residues. This protein shares a high degree of similarity with other oxytrichid actins, and a relatively low similarity with actins from other eukaryotes. Comparative analyses of sequences indicated that most of the amino acid substitutions in hypotrich actins are found in surface loops, while the core structures are well-conserved. The sites that interact with DNase I and several regions involved in actin-actin contact have diverged considerably in hypotrich actins, while nucleotide-binding sites are the best-conserved interaction motif.

Notes: An isolate of Sarcocystis neurona. (SN6) was obtained from the spinal cord of a horse from Oregon with neurologic signs. The parasite was isolated in cultures of bovine monocytes and equine spleen cells. The parasite divided by endopolygeny and completed at least one asexual cycle in cell cultures in three days. Two gamma interferon knockout mice inoculated with cell culture-derived merozoites became ill 35 d later and S. neurona schizonts and merozoites were found in encephalitic lesions. The parasite in tissue sections of mice reacted with S. neurona-specific antibodies and S. neurona was reisolated from the brain of knockout mice.

Notes: Passage through the cell cycle in eukaryotes requires the successive activation of different cyclin-dependent protein kinases. Here, we describe the identification and characterization of a novel class of cyclin-dependent protein kinase, termed Cdk2, in the ciliate Paramecium tetraurelia. It is 301 amino acids long, 7 amino acids shorter than Cdk1, the CDK that is associated with macronuclear DNA synthesis. All the catalytic domains typical of protein kinases can be located within the sequence and putative regulatory phosphorylation sites equivalent to Thr14, Tyr15, and Thr161 in human CDK1 are also conserved. The‘PSTAIRE’region characteristic of most CDKs is perfectly conserved. Cdk2 shares only 48% homology to Cdk1 at the amino acid level, suggesting that the evolutionary separation of Cdk1 and Cdk2 is ancient, and implying that they have different roles in cell cycle regulation. Like Cdk1, Cdk2 does not bind to yeast pl3suc1, even though it has better conservation of pl3suc1 binding sites than Cdk1 does. The Cdk2 protein level is relatively constant throughout the vegetative cell cycle. Cdk2 exhibits kinase activity towards bovine histone H1 in vitro with the maximal level late in the cell cycle, suggesting it may be involved in the regulation of cytokinesis. Our results further support the view that an analogue of the cyclin-dependent kinase cell cycle regulatory system like that of yeast and higher eukaryotic cells operates in Paramecium and that a family of cyclin-dependent kinases may control different aspects of the Paramecium cell cycle.

Notes: The interphase cells of the hypotrich ciliate Paraurostyla weissei possess a complex fibrillar system surrounding basal bodies in the compound ciliary assemblages, cirri and membranelles. During replacement of the ciliature at cell division, transient filaments precede and accompany the development of ciliary primordia and participate in the formation of the fission furrow. Both fibrillar systems are recognized by monoclonal antibody FXXXIX 12G9. We studied regeneration of cellular fragments after transection employing the mAb 12G9 and found a new cytoskeletal structure involved in healing of the excisional wound. The healing filament is formed at the wound edge, distally and in connection with the bases of cirri closest to the wound. It is visible 5 min after transection. Concomitant with development of new ciliary primordia, the healing filament shrinks and finally disappears together with other transient fibers formed in this process. Ultrastructural analysis of immunolabeled regenerating cells revealed that structures recognized by mAbl2G9 contain fine filaments whose packing and arrangement depends on accompanying cytoplasmic elements and the developmental status of a fragment. Assembly of the healing fiber does not depend on microtubules and microfilaments since it develops in cellular fragments exposed to cold, nocodazole, and Cytochalasin D. On Western blots of whole cell and cytoskeletal extracts of P. weissei the 12G9 antibody identified one protein band whose molecular weight corresponds to 60 kDa.

Notes: A gene encoding an α-tubulin of Cryptosporidium parvum was isolated and characterized. It had no introns, and encoded a 441-amino acid protein whose predicted ORF represented a typical α-tubulin protein with a MW of 50.5 kDa. This tubulin had an amino acid sequence similarity with Apicomplexa Plasmodium falciparum and Toxoplasma gondii higher than 88% and shared a number of conserved motifs.

Notes: Small subunit rRNA sequence data were generated for 27 strains of cyanobacteria and incorporated into a phylogenetic analysis of 1,377 aligned sequence positions from a diverse sampling of 53 cyanobacteria and 10 photosynthetic plastids. Tree inference was carried out using a maximum likelihood method with correction for site-to-site variation in evolutionary rate. Confidence in the inferred phylogenetic relationships was determined by construction of a majority-rule consensus tree based on alternative topologies not considered to be statistically significantly different from the optimal tree. The results are in agreement with earlier studies in the assignment of individual taxa to specific sequence groups. Several relationships not previously noted among sequence groups are indicated, whereas other relationships previously supported are contradicted. All plastids cluster as a strongly supported monophyletic group arising near the root of the cyanobacterial line of descent.

Notes: Plastids with two bounding membranes—as exemplified by red algae, green algae, plants, and glaucophytes—derive from primary endosymbiosis: a process involving engulfment and retention of a cyanobacterium by a phagotrophic eukaryote. Plastids with more than two bounding membranes (such as those of euglenoids, dinoflagellates, heterokonts, haptopytes, apicomplexa, cryptomonads, and chlorarachniophytes) probbly arose by secondary endosymbiosis. in which a eukaryotic alga (itself the product of primary endosymbiosis) was engulfed and retained by a phagotroph. Secondary endosymbiosis transfers photosynthetic capacity into heterotrophic lineages, has apparently occurred numerous times, and has created several major eukaryotic lineages comprising upwards of 42.600 species. Plastids acquired by secondary endosymbiosis are sometimes referred to as “second-hand.” Establishment of secondary endosymbioses has involved transfer of genes from the endosymbiont nucleus to the secondary host nucleus. Limited gene transfer could initially have served to stabilise the endosymbioses, but it is clear that the transfer process has been extensive, leading in many cases to the complete disappearance of the endosymbiont nucleus. One consequence of these gene transfers is that gene products required in the plastid must be targeted into the organelle across multiple membranes: at least three for stromal proteins in euglenoids and dinoflagellates, and across five membranes in the case of thylakoid lumen proteins in plastids with four bounding membranes. Evolution of such targeting mechanisms was obviously a key step in the successful establishment of each different secondary endosymbiosis. Analysis of targeted proteins in the various organisms now suggests that a similar system is used by each group. However, rather than interpreting this similarity as evidence of an homologous origin, I believe that targeting has evolved convergently by combining and recycling existing protein trafficking mechanisms already existing in the endosymbiont and host. Indeed, by analyzing the multiple motifs in targeting sequences of some genes it is possible to infer that they originated in the plastid genome, transferred from there into the primary host nucleus, and subsequently moved into the secondary host nucleus. Thus, each step of the targeting process in “secondhand” plastids recapitulates the gene's previous intracellular transfers.

Notes: Our understanding of the great diversity and novelty of dinoflagellate feeding ecology (summarized in Fig. 1) is progressing rapidly after a slow, erratic beginning a century ago. This advance has not been based primarily on technical innovations. Rather, basic microscopy of wild material, like that employed a century ago, has continued to yield many important insights. Indeed, several of the most exciting discoveries (including the pallium of thecate heterotrophs and the ingestion of ciliates and dinoflagellates by both naked and thecate dinoflagellates) are actually rediscoveries that have expanded upon reports published sixty to ninety years earlier. The elucidation of feeding strategies among thecate species has advanced particularly rapidly, with the recent addition of over a dozen widespread and important thecate genera to the rank of phagotroph, leading to a significant paradigm shift: the theca can no longer be considered an insurmountable or even a significant barrier to phagotrophy. This research is now developing from a descriptive and anecdotal stage to an experimental and quantitative stage, involving analysis of rates, ecological roles, and survival strategies: however, important new descriptions are still emerging. It has been shown that dinoflagellates, despite having relatively low densities and growth rates compared to ciliates, are ecologically significant. They can even compete with and prey upon microcrustacean grazers. Among many future advances in this field, perhaps the most significant will be an understanding of nano-sized dinoflagellates, particularly “Gymnodinium” and “Gyrodinium” spp. that have already been shown to play major roles in marine food webs. This development awaits a massive taxonomic overhaul of this diverse, polyphyletic assemblage. In addition, the feeding habits of well characterized thecate taxa still await characterization.

Notes: Three types of feeding mechanisms are known in dinoflagellates: pallium feeding, tube feeding, and direct engulfment. Pallium feeding has only been described for heterotrophic thecate species (Protoperidinium, Diplopsalis group). Tube feeding is commonly found among both naked and thecate species of mixotrophic and heterotrophic dinoflagellates (e.g. Amphidinium, Dinophysis, Gyrodinium, Peridiniopsis). Direct engulfment is mainly found among naked species (e.g. Gymnodinium, Gyrodinium, Noctiluca): recently, however, some thecate species have been shown to use this feeding mechanism as well. Feeding behavior in dinoflagellates involves several steps prior to actual ingestion, including precapture, capture, and prey manipulation. As feeding mechanisms allow the ingestion of relatively large prey or parts thereof, dinoflagellates are regarded as raptorial feeders. While prey size plays an important role in the ability of dinoflagellates to ingest food, this alone cannot explain observed prey preferences. Some dinoflagellate species can be very selective in their choice of prey, while others show a remarkable versatility.

Notes: Several genera of marine dinoflagellates contain species that have evolved parasitic life styles. Dinoflagellate infections have been reported for a wide range of host organisms including sarcodines. ciliates, free-living dinoflagellates, various invertebrates, and a few vertebrates. Some dinoflagellates even parasitize other parasitic dinoflagellates. Most species are obligately parasitic and rely on heterotrophy as their sole means of nutrition; however, some are mixotrophic, as they possess chloroplasts during part or all of their life cycle. Many are ectoparasites that use highly specialized structures to attach to their host and feed, while others are intracellular parasites that feed by osmotrophy. Parasitic dinoflagellates often have adverse effects on their host that can lead to reproductive castration or death. The ecological importance of parasitic dinoflagellates is particularly evident during epidemic outbreaks that cause mass mortality of host organisms. Species that infect fish can pose threats to aquaculture. while other species can make commercially important crustacea unpalatable. In the planktonic realm, parasitic dinoflagellates influence the structure and function of the microbial food web. They compete with copepods and other grazers by utilizing ciliates as hosts and can stimulate rapid recycling of nutrients by causing the decline of toxic and non-toxic red tides.

Notes: Using transmission electron microscopy, fluorescence microscopy, immuno-electron microscopy, and biochemical techniques such as 2-D electrophoresis and immunoblotting, actin was found in all biological stages of the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi.

Notes: Microsporidia are obligate intracellular parasites that are increasingly recognized as a cause of opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi has been identified in humans with AIDS and infects a wide range of mammalian hosts. Little is known about the metabolic processes that regulate growth and replication of microsporidia. Examination of the individual stages of development will facilitate such studies and reveal possible targets for drug therapy. The purpose of this study was to fractionate and purify stages of the microsporidian life cycle. Encephalitozoon cuniculi were cultured in RK-13 cells. The tissue supernatants containing multiple parasite stages, empty microsporidial husks and host cell debris were collected, washed, and subjected to differential centrifugation in 80% stock isotonic Percoll®. Transmission electron microscopy and SDS-polyacrylamide gel electrophoresis were used to compare the content and purity of each fraction. Mature spores formed a band at a density of approximately 1.138 g/ml. Sporoblasts were found at densities between 1.102 g/ml and 1.119 g/ml. A mixture of sporonts, sporoblasts, microsporidial husks, and cell debris remained at the top of the gradient and additional centrifugation in 30% and 50% Percoll® resulted in separation of these stages. These results represent the first step toward fractionating stages of microsporidia infecting humans.

Notes: In anaerobes, decarboxylation of pyruvate is executed by the enzyme pyruvate: ferredoxin oxidoreductase, which donates electrons to ferredoxin. The pyruvate:ferredoxin oxidoreductase and its homologues utilise many alternative substrates in bacterial anaerobes. The pyruvate:ferredoxin oxidoreductase from anaerobic protozoa, such as Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica have retained this diversity in usage of alternative keto acids for energy production utilising a wide variety of substrates. In addition to this flexibility, both T. vaginalis and G. duodenalis have alternative enzymes that are active in metronidazole-resistant parasites and that do not necessarily involve donation of electrons to characterized ferredoxins. Giardia duodenalis has two oxoacid oxidoreductases, including pyruvate:ferredoxin oxidoreductase and T. vaginalis has at least three. These alternative oxoacid oxidoreductases apparently do not share homology with the characterized pyruvate:ferredoxin oxidoreductase in either organism. Independently, both G. duodenalis and T. vaginalis have retained alternative oxoacid oxidoreductase activities that are clearly important for the survival of these parasitic protists.

Notes: . The characteristic grain size distributions observed in the test walls of agglutinated foraminifera can be replicated by numerical simulations. the logistics employed by the model parallel those seen in foraminifera during culturing experiments of test construction. Both fractal and log-normal grain distributions are generated by a simple space-filling algorithm. However, to generate the specific grain distributions observed in foramimferan tests a strong preference for selecting larger grains from the available sediment must be effected. the exclusion of smaller grains, during selection, produces log-normal grain distributions similar to those previously observed. These and similar modelling techniques may provide valuable insights into the mechanisms of shell construction employed by protists.

Notes: . The effect of a monoclonal antibody (1209-C2) elicited against sporozoite refractile-body antigen on invasion of cultured baby hamster kidney cells by avian Eimeria species was examined in vitro. Pretreatment of sporozoites with 1209-C2 for 45 min before inoculation into cultures or simultaneous introduction of sporozoites and 1209-C2 into cultures had no significant effect on invasion. However, pretreatment of cultures for 45 min with 1209-C2 (also with media from other cloned 1209 cell lines) significantly inhibited invasion by sporozoites of Eimeria tenella and E. adenoeides. Pretreatment of cultures with 2 unrelated monoclonal antibodies with the same isotype as 1209-C2 did not inhibit invasion by E. tenella. There was a significant correlation between time of exposure of the cultures to 1209-C2 and invasion (r = -0.80924; p = 0.0001), with inhibition of invasion occurring after 20 min exposure, but not after 10 min. There was also a significant correlation between the titer of 1209-C2 and invasion (r = 0.62291; p = 0.0305). Monoclonal antibody 1209-C2 cross-reacted with epitopes of baby hamster kidney cells by both immunofluorescence and Western blot. the fluorescent labeling of the cells differed according to the fixative that was used. In formalin-fixed cultures labeled with 1209-C2, fluorescent foci were distributed over the entire cell; after methanol fixation, 1209-C2 reacted with only discrete foci in the nucleus. On Western blots of sporozoites, 1209-C2 reacted with antigens having molecular sizes of ∼ 8, 17, 23, 30, and 45-60 kDa, and with several minor bands. On baby hamster kidney cells, the antibody reacted primarily with bands of ∼ 30, 45-60. and slightly with other bands. the data suggest that interactions among similar molecules in the sporozoites and host cells may play a role in cellular invasion.

Notes: . The behavior of populations of Uronychia setigera (Ciliata, Hypotrichida) exposed to water currents flowing at increasing velocities (300, 400, 500, 900, 1,700 μm/s) was analyzed using two techniques: 1) the ethogram and 2) the numerical indices recently proposed to measure the development in space and time of tracks of ciliates. Beyond a certain threshold value of the water velocity (˜ 300 μm/s), this species shows a definite positive rheotaxis, only if it moves in a more or less direct contact with the substrate. No rheotactic swimming ever occurs. Rheotaxis is a gradual, adaptive behavior: the higher the velocity of the current, the stronger the degree of the rheotactic response, as demonstrated by the increasing significance of the polar distribution of the tracks. Beyond 500 μm/s the water flow is so strong that it affects the locomotion of U. setigera continuously and strongly inducing this species to perform a new behavioral pattern, the Fast Backward Bidimensional Swimming. Under stressing water currents it reacts at first by creeping along straighter trajectories and then with faster locomotion, in such a way that its reaction is to a certain extent proportional to the drag of the currents. the rheotaxis of U. setigera is discussed as an adaptive response.

Notes: . Opisthonecta matiensis n. sp. was isolated from the inlet water of a wastewater treatment plant near Madrid. Spain, and studied in vivo, with silver methods, and using electronic and indirect immunofluorescence microscopy. This new species shows an amphora-like cell shape and has a size of 45-73 μm (x̄ 58.2) × 25-40 μm (x̄ 31.3). the oral infraciliature is formed by one haplokinety, three polykineties, and a short row of kinetosomes (epistomial membrane). the aboral infraciliature is made up of the trochal band and the scopula. From the trochal band arise three fibrillar systems: oral fibers, aboral fibers, and oblique fibers. the myoneme system is composed of a delicate peristomial ring, longitudinal branched fibers that reach the trochal band and of radial fibers extending from the scopula to the trochal band. the silverline system consists of an average of 147 lines. This new species is separated from other known forms by its smaller size, the presence of one single vacuole, and its higher number of silverlines.

Notes: . The parasites Babesia canis and Babesia gibsoni (phylum Apicomplexa) are responsible for canine babesiosis throughout the world. Babesia canis was previously described as a group of three biologically different subspecies, namely B. canis canis. B. canis vogeli, and B. canis rossi. We report partial sequences of small subunit ribosomal RNA gene (ssu-rDNA) of each subspecies amplified in vitro with primers derived from a semi-conserved region of the ssu-rDNA genes in other Babesia species. the polymerase chain reaction combined with a restriction fragment length polymorphism analysis, using Hinfl and Taql restriction enzymes, confirmed the separation of B. canis into three subspecies. These sequences were compared with previously published sequences of other Babesia species. A phylogenetic approach showed that the three subspecies of B. canis belong to the clade of Babesia species sensu stricto where B. canis canis clusters with B. canis rossi whereas B. canis vogeli might form a monophyletic group with the cluster B. divergens and B. odocoilei. Our results show that the three subspecies of B. canis can readily be differentiated at the molecular level and suggest that they might be considered as true species.

Notes: . Metacystis truncata, a prostomatid loricate ciliate, was found attached to Thalassia tesudinum, a dominant seagrass of the coral reef lagoons of Veracruz, Mexico. the morphology of the ciliate and its lorica were studied in living and stained specimens and in those prepared for scanning electron microscopy. the lorica is cylindrical (64.6-115μm) with a well-developed anterior neck and a sac reinforced by 15-26 rings. Cell body size is variable (19-91 μm live), from ovoid to elongate: the terminal protruding vacuole is conspicuous in small and medium-sized individuals; the macronucleus is terminal in the elongate forms, or located near the mid-body in the ovoid ones. This marine prostomatid colonizes both natural and artificial substrates such as narrow polyethylene strips placed in an aquarium. the highest density was found in the summer. the present study represents the first description of the lorica of M. truncata and the first record on T. testudinum shoots.

Notes: Trypanosomes possess a single flagellum that is attached to their cell body via the flagellum attachment zone (FAZ). The FAZ is composed of two structures: a cytoplasmic filament complex and four microtubules situated next to it. There is a complex transmembrane crosslinking of this FAZ to the paraflagellar rod (PFR) and axoneme within the flagellum. We have partially purified the FAZ complex and have produced monoclonal antibodies both against the FAZ and the paraflagellar rod. The two antibodies against the FAZ (L3B2 and L6B3) recognise the cytoplasmic filament in immunofluorescence and in immunoelectron microscopy. On Western blot, they detect a doublet of high molecular weight (M, 200,000). Two anti-PFR antibodies (L13D6 and L8C4) recognise the paraflagellar rod in immunofluorescence, but show a difference on Western blot: L13D6 recognises both major PFR proteins, whereas L8C4 is specific for only one of them. Using these new antibodies we have shown that although the growth of both cytoplasmic FAZ filament and external PFR are related, their growth initiates at different time points during the cell cycle and the two structures elongate at distinct rates.

Notes: The discovery of a non-photosynthetic plastid genome in Plasmodium falciparum and other apicomplexans has provided a new drug target, but the evolutionary origin of the plastid has been muddled by the lack of characters, that typically define major plastid lineages. To clarify the ancestry of the plastid, we undertook a comprehensive analysis of all genomic characters shared by completely sequenced plastid genomes. Cladistic analysis of the pattern of plastid gene loss and gene rearrangements suggests that the apicomplexan plastid is derived from an ancestor outside of the green plastid lineage. Phylogenetic analysis of primary sequence data (DNA and amino acid characters) produces results that are generally independent of the analytical method, but similar genes (i.e. rpoB and rpoC) give similar topologies. The conflicting phylogenies in primary sequence data sets make it difficult to determine the exact origin of the apicomplexan plastid and the apparent artifactual association of apicomplexan and euglenoid sequences suggests that DNA sequence data may be an inappropriate set of characters to address this phylogenetic question. At present we cannot reject our null hypothesis that the apicomplexan plastid is derived from a shared common ancestor between apicomplexans and dinoflagellates. During the analysis, we noticed that the Plasmodium tRNA-Met is probably tRNA-fMet and the tRNA-fMet is probably tRNA-Ile. We suggest that P. falciparum has lost the elongator type tRNA-Met and that similar to metazoan mitochondria there is only one species of methionine tRNA. In P. falciparum, this has been accomplished by recruiting the fMet-type tRNA to dually function in initiation and elongation. The tRNA-Ile has an unusual stem-loop in the variable region. The insertion in this region appears to have occurred after the primary origin of the plastid and further supports the monophyletic ancestory of plastids.

Notes: Heterotrophic dinoflagellates are ubiquitous and often abundant protists in marine environments. Recently, several novel predator-prey relationships between heterotrophic dinoflagellates and other planktonic organisms have been discovered and shown to have diverse ecological roles. Heterotrophic dinoflagellates are predators on a wide array of prey items, including phytoplankton, copepod eggs, and early naupliar stages. They are in turn important prey for some metazoa. Some heterotrophic dinoflagellates are predators of and simultaneously prey for other dinoflagellates. These newly discovered predator-prey relationships may influence our conventional view of energy flow and carbon cycling in the marine planktonic community.

Notes: Mixotrophy, used herein for the combination of phototrophy and phagotrophy, is widespread among dinoflagellates. It occurs among most, perhaps all, of the extant orders, including the Prorocentrales, Dinophysiales. Gymnodiniales, Noctilucales, Gonyaulacales, Peridiniales, Blastodiniales. Phytodiniales, and Dinamoebales. Many cases of mixotrophy among dinoflagellates are probably undocumented. Primarily photosynthetic dinoflagellates with their “own” plastids can often supplement their nutrition by preying on other cells. Some primarily phagotrophic species are photosynthetic due to the presence of kleptochloroplasts or algal endosymbionts. Some parasitic dinoflagellates have plastids and are probably mixotrophic. For most mixotrophic dinoflagellates, the relative importance of photosynthesis, uptake of dissolved inorganic nutrients, and feeding are unknown. However, it is apparent that mixotrophy has different functions in different physiological types of dinoflagellates. Data on the simultaneous regulation of photosynthesis, assimilation of dissolved inorganic and organic nutrients, and phagotophy by environmental parameters (irradiance. availablity of dissolved nutrients, availability of prey) and by life history events are needed in order to understand the diverse roles of mixotrophy in dinoflagellates.

Notes: Paramecium nephridiatum Gelei. 1925, was rediscovered. It is a euryhaline brackish-water species that morphologically resembles Paramecium woodruffi. but with multiple contractile vacuole pores. The general morphology, morphometry. and random amplified polymorphic DNA (RAPD) fingerprint patterns are presented for a number of the stocks collected around the world.

Notes: The small-subunit rRNA sequence of a species of Amoebophrya infecting Gymnodinium sanguineum in Chesapeake Bay was obtained and compared to the small subunit rRNA sequences of other protists. Phylogenetic trees constructed with the new sequence place Amoebophrya between the remaining dinoflagellates and other protists.

Notes: The morphology and morphogenesis of Metopus hasei Sondheim, 1929 and M. inversus (Jankowski, 1964) n. comb, were investigated using live observation, silver impregnation, and scanning electron microscopy. Metopus has a spiral body organization and the ventral margin of the preoral dome bears five specialized ciliary rows, that form the so-called perizonal stripe. Division is homothetogenic, occurs in freely motile (i. e. non-encysted) condition, and includes a partial reorganization of the parental oral apparatus. During division, the complicated cell shape becomes ellipsoidal and all ciliary rows arrange meridionally. Stomatogenesis is entirely somatic (≅ pleurotelokinetal) and commences with the formation of kinetofragments in some dorsolateral kineties. The fragments become the opisthe's adoral membranelles, while the paroral membrane is generated by the left two perizonal ciliary rows, which proliferate kinetids intrakinetally. The perizonal stripe of the opisthe is generated by the three right parental perizonal kineties, which divide, and by two dorsolateral ciliary rows, which are added. The morphogenetic processes, especially the unique mode of formation of the paroral membrane, are used to define the order Metopida Jankowski, 1980 n. stat. more properly. The ontogenetic, ultrastructural, and sequence data available give no clear indication about metopid phylogeny, but definitely exclude metopids from the classical heterotrichs, with which they were classified for more than 100 years. Accordingly, we place the Metopida as incertae sedis in the subphylum Intramac-ronucleata Lynn, 1996.

Notes: The euglenoids are an ancient and extremely diverse lineage of eukaryotic flagellates with unclear relationships among taxa. Synapomorphies for the euglenoids include a surface pellicle and a closed mitosis with a series of separate sub-spindles. The taxonomy currently in use is inconsistent with the available data and needs revision. Most euglenoid phylogenies are largely intuitive reconstructions based on a limited number of morphological characters. Therefore, we have added molecular characters from the Small Subunit (SSU) rDNA to generate an overall phylogenetic framework for the euglenoids. SSU rDNA sequences from photosynthetic, osmotrophic, and phagotrophic euglenoids were aligned based on secondary structure. Phylogenetic analysis using the conserved areas of the sequence was performed using parsimony, maximum likelihood, and distance methods. Trees derived using different criteria are in agreement. The euglenoids form a distinct monophyletic clade with phagotrophic members diverging prior to the phototrophic and osmotrophic members. Among photosynthetic members, the biflagellate form diverged prior to the uniflagellate form. Additionally, the genus Euglena appears to be paraphyletic, with osmotrophic taxa, such as Astasia and Khawkinea, diverging independently within the clade containing the photosynthetic genus Euglena.

Notes: . In recent years, the amount of molecular sequencing data from Tetrahymena thermophila has dramatically increased. We analyzed G + C content, codon usage, initiator codon context and stop codon sites in the extremely A + T rich genome of this ciliate. Average G + C content was 38% for protein coding regions. 21% for 5′ non-coding sequences, 19% for 3′ non-coding sequences, 15% for introns, 19% for micronuclear limited sequences and 17% for macronuclear retained sequences flanking micronuclear specific regions. the 75 available T. thermophila protein coding sequences favored codons ending in T and, where possible, avoided those with G in the third position. Highly expressed genes were relatively G + C-rich and exhibited an extremely biased pattern of codon usage while developmentally regulated genes were more A + T-rich and showed less codon usage bias. Regions immediately preceding Tetrahymena translation initiator codons were generally A-rich. For the 60 stop codons examined, the frequency of G in the end + 1 site was much higher than expected whereas C never occupied this position.