bluuebunny/crossref_metadata_2025 huggingface dataset:
Possible artificial introductions of freshwater peritrich ciliates and their implications for assessment of protistan biodiversity
Authors: CLAMP JOHN C.
Published: April 2005

The planktonic, sessiline peritrichs Epicarchesium pectinatum (Zacharias 1897) and Pelagovorticella natans (Fauré‐Fremiet 1924) were collected from different tributaries of the St. Lucie River, Florida. Epistylis procumbens Zacharias, 1897, another planktonic sessiline, was collected near the mouth of the Kissimmee River, Florida. Epicarchesium pectinatum and P. natans were not seen again in the St. Lucie drainage, probably because prolonged drought had increased the salinity of habitats drastically. Epistylis procumbens was absent from other, nearby sections of the Kissimmee River and nearby lakes and streams. Efforts to collect the three species from other bodies of water in Florida and from lakes and ponds in central New York met with no success; however, P. natans was found in two of the many localities sampled in North Carolina, an artificial pond and a natural lake. Pelagovorticella natans was found on only one of several visits to the latter locality. The three species occurred in habitats that were either within or near populated areas and had been extensively modified. This and the disjunct, ephemeral distributions of the peritrichs suggest that they may have been introduced artificially. Artificial introduction of ciliates or other protists is poorly documented but probable, considering their small size and consequent ease of transportation, and peritrichs should be especially suspect because many of them attach to aquatic plants. In the end, research on biodiversity of free‐living protists must include the possibility of artificial introductions and develop methodologies to separate them from natural occurrences or else be reduced to a meaningless exercise.
The range of protists in Mono Lake, a hypersaline soda lake in the eastern sierras
Authors: DAVIDSON LLOYD A. , DAVIDSON ARDEN E.
Published: April 2005

In spite of the controversies surrounding the environmental deterioration of Mono Lake, and the significant amount of research being done on this remarkably productive body of water, nobody has ever attempted to survey the protists that live beneath its surface. Beginning in July of 2003, we have been trying to remedy this situation and we spent 2 months there studying the protistan species that reside in its waters and since then have been receiving regular sample collections for analysis. Based on the metazoan eukaryotes that live in this lake, which include only Artemia monica, Ephydra hians, rare rotifers and a nematode, we were warned to expect the lake to be depauperate of protists as well. However, we are happy to report that, while not as rich in species as most freshwater lakes, Mono Lake does support a robust population of phagotrophic and heterotrophic protozoa in addition to its many species of unicellular algae, including diatoms. It contains ciliate species representing at least the genera Cyclidium, Frontonia, Lacrymaria, Litonotus, Euplotes and Aspidisca, and the stalked suctorian Tokophrya. Additionally, there are species representing the heliozoa, helioflagellates, choanoflagellates, Pseudobodo, Cercomonas and Chilomastix, among other flagellates. The gymnamoebae are represented by Vanella, Nuclearia, Mayorella and Hartmanella, and, from the Heterolobosea, probable Vahlkampfia and Gruberella species. Micrographs of many of these species will be presented. Work on these species is still very much in its preliminary stages and a more detailed analysis and identification of the protists found in this extreme environment is advancing.

This work was partially supported by an NSF grant (MCB 99‐77901) to Robert Jellison.
Protozoology's lost legacy
Authors: DOLAN M. F.
Published: April 2005

A review of the contents of the Journal of Protozoology/Journal of Eukaryotic Microbiology from its founding in 1954 to the present was undertaken to determine if the type specimens of new taxa named in the journal have been deposited in museum collections. Type specimens were deposited in museum collections for 36% of these new species. No information was given on the designation of type specimens or the deposition of specimens for 64% of the taxa named, mostly prior to 1980. Type specimen designations have improved greatly in recent years. All species described in the journal in the last 5 years have been deposited as type specimens or type cultures. Linda Cole's Catalog of Type Specimens in the International Protozoan Type Collection (1994) at the National Museum of Natural History was cross‐checked, and a final list of the missing organisms, grouped by phylum, was prepared. The Harold Kirby and L. R. Cleveland collections at the American Museum of Natural History are examples of how valuable the resource protozoan type specimen collections can be to researchers. Neither of these researchers had made plans to deposit their specimens in a museum. Their collections were saved only by the assistance of their students and colleagues. The author would like to coordinate an effort to find missing type specimens and facilitate their deposition in suitable museum collections.
A brief guide to identifying bacteria in the field, for protistologists
Authors: DYER BETSEY DEXTER
Published: April 2005

Bacteria are notably the most abundant and most diverse of all organisms but also the most easily overlooked and even ignored. Yet, almost all of the major groups of bacteria are identifiable in the field without a microscope. Odors, textures, colorful slimes, mats, and scums are among the bacterial field marks that provide clues as to which microbes are present. The rules‐of‐thumb and tricks‐of‐the trade for identification are often passed down to field microbiologists through graduate schools, but are seldom published for general use in other fields. Protistologists may find this brief introduction to bacterial field marks helpful for placing their own analyses and interpretations of habitats and communities into a greater context. Further information and details may be found in A Field Guide to Bacteria (Cornell 2003).
Modulation of proteins in Naegleria fowleri amebae by bacteria
Authors: FRITZINGER ANGELA E. , MARCIANO‐CABRAL FRANCINE
Published: April 2005

Naegleria fowleri are free‐living amebae found in soil and freshwater habitats that cause a fatal disease in humans called Primary Amoebic Meningoencephalitis. In the natural environment, amebae feed on bacteria. In the infected host, the amebae lyse and ingest nerve tissue. Proteins that lyse and degrade bacteria and mammalian cells have been described and are termed as naegleriapores or pore‐forming proteins (PFP). In order to prevent autolysis from PFPs, Naegleria may have developed mechanisms that enable the amebae to survive in the presence of cytolytic molecules. Recently, we have established that N. fowleri express a “CD59‐like” surface protein, but the function of this protein in amebae has not been elucidated. In mammalian cells, CD59 is a complement–regulatory protein that inhibits complement‐mediated lysis of cells expressing this protein. In the present study, expression of the “CD59‐like” protein in response to bacteria and bacterial toxins was investigated. Co‐culture of N. fowleri with log phase Escherichia coli or Pseudomonas aeruginosa resulted in differential expression of the “CD59‐like” protein. Treatment of the amebae with bacterial toxin also affected expression of the protein. Scanning and transmission electron microscopy demonstrated morphological changes in N. fowleri following co‐incubation with the bacteria. Under all conditions examined, the amebae remained intact and viable. The results of our study implicate a possible protective role of the “CD59‐like” protein in response to bacterial predators and bacterial toxins.
The hydrogenosomes of the chytrid fungus Neocallimastix frontalis—Characterizing the functions of an anaerobic cousin of mitochondria through proteome analysis
Authors: GELIUS‐DIETRICH GABRIEL , HENZE KATRIN
Published: April 2005

Hydrogenosomes are anaerobic forms of mitochondria in at least four independent groups of anaerobic eukaryotes, the parabasalids, the heterolobosea, the ciliates and the anaerobic chytridiomycetes. Their hydrogenosomes generate ATP from the fermentative decomposition of pyruvate but lack the hallmark pathways of mitochondrial metabolism, the TCA cycle and membrane‐associated electron transport. They also, with one exception, do not contain their own genome. We are analysing the proteome of the hydrogenosomes from the chytridiomycete Neocallimastix frontalis through 2D electrophoresis and tandem mass spectroscopy to assess the functional capacities of these organelles and to further characterize their relationship with mitochondria.
Two putative peroxidases of bacterial origin in the hydrogenosomes of the anaerobic protozoan Trichomonas vaginalis
Authors: AHR SIMONE , HENZE KATRIN
Published: April 2005

Trichomonas vaginalis is an anaerobic protozoan parasite of the human urogenital tract. It lacks functional mitochondria but contains hydrogenosomes, anaerobic forms of mitochondria that generate ATP from the fermentative degradation of pyruvate. To determine the similarities and differences between hydrogenosomes and mitochondria, we are characterizing the proteome of purified hydrogenosomes from T. vaginalis by 2D electrophoresis and ESI‐Q‐TOF MS/MS. In the process, we have identified two proteins of bacterial origin that may function as peroxidases and this may be part of the answer to the unsolved question of how hydrogensomes in T. vaginalis are protected against the detrimental effects of oxygen: rubrerythrin is a non‐heme iron protein found predominantly in anaerobic bacteria that can reduce H2O2 and was shown to complement catalase‐deficient E. coli mutants. Among eukaryotes we have found only one further rubrerythrin homologue in the anaerobic, amitochondriate amoeba Entamoeba histolytica. Peroxiredoxins are ubiquitous peroxidases using thiols to reduce alkyl hydroperoxides. T. vaginalis hydrogenosomal thiol peroxidase (Tpx) shows 65% homology to bacterial 20‐kDa Tpx. These peroxidases contain three highly conserved Cys residues, Cys62, Cys82, and Cys95, and belong to a peroxiredoxin subfamily that previously contained eubacterial sequences only. The finding of two putative bacterial‐type peroxidases in T. vaginalis hydrogenosomes suggests that proteins involved in peroxide detoxification in these anaerobic organelles may have been acquired laterally from eubacteria.
The shell or the kernel: biodiversity estimates of benthic foraminifera
Authors: HABURA ANDREA , HANES STEVEN D. , ALEXANDER STEPHEN P. , BOWSER SAMUEL S.
Published: April 2005

Estimates of protist biodiversity and distribution have until recently relied on the morphological identification of individual cells. The advent of environmental‐DNA‐based surveys of these protist communities has broadened our understanding of species richness and diversity. A “total‐evidence biodiversity” approach gives two benefits. First, it allows “back‐comparison” to previous studies of ecosystems, permitting estimates of the amount of cryptic diversity in different sampling regimes (and by extension, the fossil record). Second, it will provide the best possible estimate of the total species richness of current study sites. Here, we use a well‐studied benthic protist community, the foraminiferal assemblage of McMurdo Sound, Antarctica, to determine the effective parameters of both detection methods. We find that molecular‐based techniques detect well‐characterized calcareous species at depths too great to allow calcification, suggesting that these forms may be able to survive without their distinctive tests. “Fragile” taxa also appear to be better represented in molecular surveys than in morphological ones. The strengths of a combined morphological–molecular approach are demonstrated by an assessment of the distribution of two species in the genus Notodendrodes; morphological detection excels in the identification of distinctive, patchily distributed adults, whereas molecular detection with taxon‐specific probes enabled identification in less favorable sampling conditions. Such a “broad‐plus‐deep” approach appears to maximize detection efficiency in this Antarctic setting.

Work supported by NSF OPP0003639.
Characterization of the fenestrin, a cytoskeletal protein involved in Tetrahymena cell polarity and of its coding gene
Authors: JOACHIMIAK EWA , SIKORA JACEK , KACZANOWSKA JANINA , KIERSNOWSKA MAURYLA , BARCHETTA SABRINA , MICELI CRISTINA , KACZANOWSKI ANDRZEJ
Published: April 2005

Fenestrin is a cytoskeletal protein, which participates in conjugation and cell cycle regulation of Tetrahymena by formation of specific structures during these processes. In cell cycle it appears at the anterior poles of future proter and opisth cells. By 2‐D electrophoresis, we found that in starved T. thermophila cells fenestrin appears in four isoforms that have the pH in the range of 5.2–5.5. The differences of pH of about 0.1–0.3 between isoforms could suggest that they differ by the phosphorylation state. By mass spectrometry, we identified some of the peptides derived form the enzyme digestion of fenestrin. These peptides were used for searching through T. thermophila genome, available at the site http://www.tigr.org. A nucleotide sequence coding one of the peptides was initially found. Then, by extension, the sequence of all fenestrin gene appeared, containing the other peptides identified by spectrometry. By RT‐PCR we characterized one intron in this gene. The analysis of the aminoacid sequence of the exons revealed that fenestrin is composed from at least two domains: N‐terminal leucine‐rich domain and C‐terminal glutamic acid‐rich domain. The BLAST search on general databases showed the existence of human and mouse proteins homologous to fenestrin in sequence, molecular mass and domain organization. Thus, fenestrin appears to be a protein correlated to cell polarity with an evolutionarily conserved structure. Its function should be cleared by mutational analysis and gene knock out.
Effect of the micronucleus on cellular structures in several hypotrichous ciliates
Authors: JIN LIPEI , LIU XINYING , MAO YONGZHEN
Published: April 2005

It is commonly believed that there is a sharp functional demarcation between the two types of nuclei of ciliated protozoa: the micronucleus for reorganization of the nuclear apparatus during sexual reproduction and the macronucleus for controlling somatic functions. This traditional view, however, has been critically re‐examined recently and correlative researches have shown that the micronucleus plays an essential role in the development of some ciliates. Here we report some morphological evidences about the effect of the micronucleus on cellular structures from amicronucleate cell lines of five hypotrichous ciliates (Pseudourostyla cristata, Pseudokeronopsis monilata, Paraurostyla weissei, Uroleptus piscis and Oxytricha faurei). All amicronucleate cell lines were generated by amputation and investigated by protargol staining. These observations manifest that micronucleus is indispensable to vigor and viability for P. weissei, to maintaining the integrity of adoral zone of membranelles (AZM) and/or undulating membranes (UM) for P. cristata, P. monilata and U. piscis, and to keeping regularity of cell shape as well as of the number and the structure of the macronuclei for O. faurei. These results suggest that the manifestations of the somatic function of micronucleus for different ciliates are varied and the close species might have a similar form of expression.

This work was supported by a grant from NSFC (No: 30370210) to L. P. JIN.
Molecular evidence for a single common origin of chromalveolate plastids
Authors: KEELING PATRICK J. , PATRON NICOLA J. , ROGERS MATTHEW B.
Published: April 2005

Plastids (photosynthetic organelles of plants and algae) are known to have spread between eukaryotic lineages by secondary endosymbiosis, or the uptake of a eukaryotic alga by another eukaryote, but the number of times this has taken place is controversial. This is particularly so in the case of eukaryotes with plastids derived from red algae, which are numerous and diverse. Despite their diversity, it has been suggested that all these eukaryotes share a recent common ancestor and that their plastids originated in a single endosymbiosis, the so‐called “chromalveolate hypothesis”. Here, we describe a novel molecular character that supports the chromalveolate hypothesis. Fructose‐1,6‐bisphosphate aldolase (FBA) is a glycolytic/Calvin cycle enzyme that exists as one of two non‐homologous types, class I and class II. Red algal plastid‐targeted FBA is a class I enzyme related to homologues from plants and green algae, and it would be predicted that the plastid‐targeted FBA from algae with red algal secondary endosymbionts should be related to this class I enzyme. However, we show that heterokonts, cryptomonads, haptophytes and dinoflagellates (all photosynthetic chromalveolates) encode two paralogous class II enzymes, one of which strongly supports a specific relationship between chromalveolates. Exactly where chromalveolates got their plastid‐targeted FBA genes is presently uncertain, but their common possession of this unexpected plastid characteristic is compelling evidence for their close relationship and a common origin for their plastids.
Characterization of Vorticella convallaria calcium‐binding centrin proteins
Authors: KONIOR KATARZYNA , McCUTCHEON SUZANNE M. , BUHSE HOWARD E.
Published: April 2005

The stalked ciliate, Vorticella convallaria, is a good model system to study mechanochemical motility because its contractile organelles (spasmoneme and myonemes) use a mode of contraction that differs from most other eukaryotic motile systems. Since calcium triggers this contraction, we have undertaken the molecular characterization of the calcium‐binding proteins associated with these organelles. We have isolated and identified seven unique centrin‐like cDNAs from V. convallaria. Each encodes an acidic protein of approximately 20‐kDa, containing a unique N‐terminus and four potential calcium‐binding domains. We predict that each centrin has a distinct function within the cell. To define these functions, we have initiated immunofluorescence localization studies utilizing various anti‐centrin antibodies. Western analysis indicates that each antibody recognizes a distinct protein or subset of proteins in Vorticella. Using these antibodies, we have localized centrin to various structures within the cell; myonemes, spasmoneme, and the oral apparatus. Because each of these antibodies recognizes a different protein on Westerern analysis, we conclude that a number of calcium‐binding proteins are associated with the contractile organelles. To further characterize this gene family, we have initiated immunolocalization at the ultrastructural level. This will permit subcellular localization of all Vorticella centrins and enable us to dissect the function of this multi‐gene family.
Characteristics of phosphatidylinositol synthase activity from Tetrahymena
Authors: LANSLEY TANYA A. , MOYE BRANDY R. , CHAN EDMOND R. , RYALS PHILLIP E.
Published: April 2005

Properties of phosphatidylinositol synthase (PtdIns‐synthase) from the ciliate Tetrahymena were investigated using microsomal fractions enriched for the enzyme. Optimum conditions for PtdIns‐synthase activity as assayed in a Triton X‐100/CDP‐DAG mixed micelle system with [3H] myo‐Ins as co‐substrate were a pH of 8.0 using 50 mM Tris‐HCl buffer and a temperature of 30°C. Incubation of PtdIns‐synthase at pre‐selected temperatures prior to assay at 30°C showed that the enzyme was stable for at least 20 min at 40°C. A sharp decline in activity was seen after incubation at 50°C for 20 min. Activity was stimulated by 2 mM Mg2+, and stimulated to a slightly lesser degree by 1 mM Mn2+; however, these two ions in combination were synergistic. Other metal cations (Ca, Co, Cu, Fe, Mo, Zn, and Li) at a concentration of 2 mM had no appreciable effect on activity. Under optimum assay conditions, Tetrahymena PtdIns‐synthase activity was linear over at least 20 min and was linear with increasing concentrations of the enzyme. The enzyme was capable of utilizing both [3H]scyllo‐and [3H] (1D) chiro‐Ins as substrate to form phosphatidyl‐[3H]scyllo‐and phosphatidyl‐[3H](1D) chiro‐inositol, respectively, with (1D)chiro‐inositol being the least preferred substrate. The in vitro synthesized myo‐and scyllo‐inositol‐containing phosphoinositides were susceptible to hydrolysis by Bacillus cereus phosphatidylinositol‐specific phospholipase C, but the (1D) chiro‐inositol phosphoinositide was resistant. We hypothesize that putative DAG and scyllo‐IP3 second messengers produced by the cleavage of polyphospho‐scyllo‐inositides may function in novel transmembrane signaling cascades.
Phagotrophy in chlorarachniophyte algae: implications for eukaryotic genome evolution
Authors: ARCHIBALD JOHN M.
Published: April 2005

The chlorarachniophytes are a group of marine amoeboflagellate algae that acquired photosynthesis by secondary endosymbiosis. Together with cryptomonad algae, the chlorarachniophytes are unusual in that they have retained the nucleus of their eukaryotic endosymbiont. The host component of chlorarachniophyte cells is derived from a member of the Cercozoa while the chlorarachnion endosymbiont is green algal in origin. In addition to being able to photosynthesize, chlorarachniophytes have been shown to ingest other algae and bacteria, although the exact nature of engulfment, and the frequency with which this occurs in nature is unknown. Here, I present a light micrographic study of phagotrophy in three chlorarachniophytes, Chlorarachnion reptans, Lotharella sp. 240 and Gymnochlora stellata. These organisms readily ingest a wide variety of algae, including cryptomonads, haptophytes and green algae. The mechanism of capture and ingestion involves adhesion to, fusion with, and digestion within the pseudopodia formed by chlorarachniophyte cells. The implications of phagotrophy on the chlorarachniophyte nuclear genome are discussed.
Antarctic amoebae adventures
Authors: MORAN DAWN M. , ANDERSON O. R. , DENNETT MARK R. , CARON DAVID A. , GAST REBECCA J.
Published: April 2005

Ice, slush, sediment and water samples were collected from the Ross Sea, Antarctica and enriched with a variety of inorganic and organic nutrients. All cultures were maintained at an ambient temperature of 1°C, and amoebae were observed to occur in the slush, sediment and water enrichments. Amoebae were isolated into clonal or monocultures, and their characterization was accomplished using a combination of molecular and morphological methods. Full‐length 18S ribosomal DNA sequence data indicated that seven of the isolates represented four different amoebae of the Vexilliferidae and Vannellidae families. Acquisition of 18S ribosomal sequences from the parasomes of two amoebae further confirmed their identification as Neoparamoeba species. Light microscopy, fluorescence microscopy and TEM observations were accomplished and further support the molecular data. To determine whether these amoebae represented psychrophilic strains, their temperature tolerances were tested. Cultures were inoculated at 1°C and the temperature was raised 1°C/day (to a total of 5°), and then held at that temperature for 6 days. This pattern was repeated in 5°‐increments, up to 20°C. The cultures were observed frequently for death and possible cyst formation. Three (2 types of Vannellids) out of seven amoeba cultures survived up to 20°C. Whereas, the two different Vexilliferids died at a range of temperatures between 10°C and 20°C. These data indicate that amoebae found in the Antarctic are varied in their physiological adaptation for growth at cold temperatures.
The evolutionary relationships amongst excavates: a concatened protein analysis
Authors: SIMPSON ALASTAIR G.B. , INAGAKI YUJI , ROGER ANDREW J.
Published: April 2005

Morphological studies and phylogenies of ribosomal RNA, taken together, suggest that excavate protists may be related to each other, but many of the deepest level relationships amongt these organisms remain poorly understood. We have assembled a data set of six slowly evolving nuclear‐encoded protein genes that include nine of the 10 recognized excavate groups. Maximum likelihood analyses demonstrate that diplomonads and Carpediemonas than parabasalids are related to each other. They also confirm that Trimastix is specifically related to oxymonads, forming the taxon Preaxostyla. There is strong support for a clade of Euglenozoa, Heterolobsea and jakobids, but, unexpectedly, jakobids and Heterolobosea are robustly recovered as sister taxa. Malawimonas is placed either as sister to Preaxostyla or as sister to the (Euglenozoa, Heterolobosea, jakobid) clade. The original data set strongly supports an association between the (diplomonad, Carpediemonas, parabasalid) clade and Opisthokonts. However, this grouping is not recovered when α‐tubulin is excluded from the analysis, suggesting that the signal for this relationship lies within this one protein and might be suspect. All other important nodes in the tree are, by contrast, robust to the removal of any one gene. With α‐tubulin excluded, excavates tend to form just two clades, with no strong nodes separating them. Jakobids, with their apparently ancestral bacterial‐type mitrochondrial RNA polymerase are nonetheless nested within a clade with normal phage‐type RNA polymerases, complicating any understanding of deep‐level mitochondrial evolution The position of jakobids also seriously challenges the now well‐accepted concept of a taxon Discicristata.
Genome evolution in Microsporidia
Authors: SLAMOVITS CLAUDIO H. , KEELING PATRICK J.
Published: April 2005

Microsporidia are eukaryotic intracellular parasites that evolved from fungi. However, they are highly derived and specialized, and lack several typical eukaryotic features, such as canonical mitochondria, flagella and peroxisomes. They also exhibit seemingly “primitive” traits, probably due to their extreme sequence divergence and reductive evolution, which long obscured their true phylogenetic affinity. Gene content and genomic architecture in Microsporidia are known from E. cuniculi, the only species whose complete genome has been sequenced. A genomic sequence survey of A. locustae, a second and distantly related species, carried out in our laboratory provided valuable data for many studies that improved our knowledge on the biology and evolution of Microsporidia. We present here the latest results of our survey, emphasizing comparative analyses between A. locustae and E. cuniculi in order to explore the dynamics of genome evolution and compaction in this intriguing group of parasites. We found that, in spite of their extreme rates of sequence evolution, microsporidian genome structure evolves slowly compared to other eukaryotic groups and we propose that it is a consequence of compaction. We also report the discovery and characterization of a photolyase in A. locustae that protects the spore from DNA damage. In addition, comparative analysis allows the identification of several important genes that would otherwise not be recognised due to the extreme level of sequence evolution. Finally, we compared intergenic DNA between both species aiming to understand the structure of regulatory sequences in these highly compacted genomes.
Hidden diversity in the tide pool ciliate Strombidium occulatum as revealed by mutiple molecular markers
Authors: SNOEYENBOS‐WEST OONA L.O. , PIROG KATARZYNA A. , GRIFFIN AUTUMN , MCMANUS GEORGE , COSTAS BARBARA , KATZ LAURA A.
Published: April 2005

We find tremendous genetic diversity underlying the morphological entity Strombidium occulatum. We used multi‐locus sequence data to examine molecular diversity within S. occulatum across space and time so as to explore the biogeography and biodiversity of this marine ciliate species. We are sequenceing the internally transcribed spacer regions (ITS1 and ITS2) of the SSU rDNA gene, Mitochodrial SSU and Histone H4 from S. occulatum populations collected on either side of the North Atlantic Ocean: from tide pools along the western coast of the United Kingdom and the southern coast of New England. Samples were collected over short temporal (tidal) and both small and large spatial scales in order to explore the level of intraspecific genetic variation. Analyses of our sequence data show that a high level of DNA polymorphisms and genetic heterogeneity (e.g. up to 13% divergence at the ITS locus) underlies S. occulatum, indicating the existence of extensive cryptic speciation in this ciliate. We propose that the high levels of genetic diversity in S. occulatum arose during times of population subdivision (allopatry), perhaps driven by glacial retreat and sea‐level change during the Middle Miocene (∼15 mya) in Europe.
Understanding genome complexity and protein evolution in ciliates: insights from mitchondria and nuclei
Authors: SNOEYENBOS‐WEST OONA L. O. , LASSER ELYSE , CARDILLO JESSICA C. , KATZ LAURA A.
Published: April 2005

We are exploring the evolutionary implications of genome duality in ciliates. Ciliates possess both a somatic macronucleus (MAC) and a germline micronucleus (MIC). Only the MAC is transcriptionally active, and divides by amitosis rather than mitosis and meiosis. We have previously hypothesized that genome duality is the driving force behind the heterogeneous and unusually elevated rates of amino acid substitutions we find in some protein coding genes. Does the dimorphic nature of the ciliate genome allow them to “cross the valleys in the adaptive landscape” of protein evolution? To test this hypothesis, we are analyzing additional protein genes as well as mitochondrial genes. Mitochondrial genes serve as an excellent comparison to those of the nucleus because phylogenetic information lost by the rapidly evolving nuclear genome may be retained in the mitochondria.
The alternative oxidase from Euglena gracilis
Authors: THEISSEN URSULA , HEBING PEER , MARTIN WILLIAM
Published: April 2005

The alternative oxidase (AOX) is an ubiquinol oxidase of the mitochondrial respiratory chain. AOX is an additional terminal oxidase; the electron flux to AOX branches from the classical electron‐transport chain at the level of the ubiquinone pool leading to the direct reduction of oxygen to water in a single four‐electron transfer step. O2 reduction catalyzed by AOX is not coupled to proton pumping and hence not ATP‐producing. The energy is released as heat. AOX is specifically inhibited by salicylhydroxamic acid (SHAM), but is cyanide resistant. AOX was discovered in plant thermogenic tissues and all plant species tested to date can express the alternative pathway. It was long thought that AOX is specific to higher plants, but it is now turning up in many non‐photosynthetic eukaryotes. For example the bloodstream form of the protist Trypanosoma brucei brucei that lacks functional cytochromes, limits most of its energy metabolism to glycolysis while passing the reducing equivalents produced to water via a plant‐like alternative oxidase. It has also been reported that oxidative phosphorylation in the protist Euglena gracilis is supported by an alternative respiratory pathway in which an AOX is involved. We have cloned the mitochondrial AOX from Euglena and have investigated the evolutionary history of this enzyme in prokaryotes and eukaryotes.
Not the most basal eukaryotes: challenges and pitfalls affecting the placement of Giardia and Trichomonas
Authors: WALKER GISELLE , EMBLEY T. MARTIN
Published: April 2005

Diplomonads (including Giardia) and parabasalids (including Trichomonas) have traditionally been seen as the deepest‐branching eukaryotes, on the evidence of apparently simple ultrastructural morphology, and small subunit ribosomal RNA trees. However, this position does not appear to be well supported, especially in the light of the discoveries of apparent mitochondrial remnants in Giardia and Trichomonas. We systematically re‐analysed protein data, and show here that their traditional placement may be due to a combination of factors acting on data sets (narrow taxon sampling) and on genes (rate heterogeneity and compositional heterogeneity, mutational saturation). Our data found no consistent support for either Giardia or Trichomonas being basal eukaryotes.
Ionic currents and cytoplasmic streaming in Amoeba proteus
Authors: BARRY SUSAN R. , CORSON ERICA D.
Published: April 2005

Amoeba proteus may drive a current through themselves, and this current may determine the direction of cytoplasmic streaming. Cytoplasm streams from the more contracted tail or uroid region to the extending pseudopods. The positions of the uroid and pseudopods change as the amoeba changes direction. The membrane potential of the amoeba may change with location in the cell. This voltage gradient may regulate the magnitude and direction of cytoplasmic streaming. Bingley and Thompson (1962), recorded from amoebae using intracellular microelectrodes and reported that the membrane potential of the uroid region was more negative than that of the pseudopods. In contrast, Nuccitelli et al. (1977), using a vibrating probe, observed that current entered the uroid region and exited through the pseudopods. They concluded that the uroid region was more depolarized than the pseudopods. We revisited this issue by recording membrane potential simultaneously from two regions of the amoeba and by recording ionic currents using the two‐electrode voltage clamp. Our data support the findings of Nuccitelli et al. Cytoplasm may stream from more depolarized to more hyperpolarized regions. Moreover, overshooting depolarizations of the cell may produce global cytoplasmic contractions and rounding of the amoeba.
Filling in the gaps: EST surveys of under‐represented chromalveolates
Authors: WALLER ROSS F. , PATRON NICOLA J. , KEELING PATRICK J.
Published: April 2005

The “chromalveolate hypothesis” posits the monophyly of alveolates (apicomplexans, dinoflagellates and ciliates), heterokonts, haptophytes and cryptomonads. One implication of the monophyly of chromalveolates is that the plastids in this group could share a common ancestor—derived from a single endosymbiosis. Members of all chromalveolate groups, with the exception of ciliates, possess a secondary plastid originating from a rhodophytic symbiont (although the rhodophytic origin of the apicomplexan plastid remains contentious). A single origin of this chromalveolate plastid represents the most parsimonious hypothesis for plastid acquisition. Growing support for the “chromalveolate hypothesis” has relied on a relatively small number of molecular studies to date, and a more thorough examination of this alliance has been undermined by a paucity of broad molecular data from several of its members, despite complete genome sequences from apicomplexans and ciliates. To address this deficit we have undertaken EST (Expressed Sequence Tag) surveys of several taxa, representing dinoflagellates, haptophytes and cryptomonads. Using EST data we are undertaking broad molecular comparisons of these groups through molecular phylogenies, analyses of common biochemical pathways, and studies of organelle evolution. This genome‐based approach provides a more complete view of the major chromalveolate groups, and a chance to more thoroughly test the chromalveolate hypothesis.
Developmental genome processing and protein evolution in the ciliate Chilodonella uncinata
Authors: ZUFALL REBECCA A. , KATZ LAURA A.
Published: April 2005

Ciliates are microbial eukaryotes with dual genomes, one present in the transcriptionally inactive germline micronucleus (MIC) and the other in the somatic macronucleus (MAC). In the development of the MAC from the MIC, ciliates process their genomes by chromosomal fragmentation, excision of internal excised sequences (IESs), and amplification of chromosomes. Chilodonella uncinata is in a class of ciliates, Phyllopharyngea, that undergo extensive processing to generate MACs containing thousands of gene‐sized chromosomes. Previous analyses suggest that sequences involved in this processing are highly variable among ciliate lineages. In this study, we examine cis‐acting signals involved in the elimination of IESs in C. uncinata in order to understand the phylogenetic level at which processing signals are conserved. In addition, we are testing the hypothesis that the differential selection on dual genomes in ciliates allows unusually rapid divergence among paralogs of protein coding genes.
Interactions of Acanthamoeba polyphaga with Listeria monocytogenes and MRSA
Authors: HUWS S. A. , BROWN M. R. W. , SMITH A. W.
Published: April 2005

Bacterivorous protozoa and bacteria have been in co‐existence since the origin of life. This co‐existence has led unequivocally to the evolution of many different co‐interactions. Most bacteria are ingested and digested, but many escape ingestion for various reasons. Others are ingested but evade digestion, and a few, notoriously Legionella pneumophila, even have the capacity of multiplying within the protozoan host. The aims of this study were to elucidate the interactions of various multi‐drug‐resistant Staphylococcus aureus (MRSA) strains, Listeria monocytogenes sv4b, and Escherichia coli K12 with the amoeba, Acanthamoeba polyphaga. To evaluate the interactions, we set up co‐cultures in Neffs' amoebic saline, at a multiplicity of invasion (MOI) of 1:100 of amoeba to bacteria, and a temperature of 37°C, although the effects of MOI and temperature were also assessed. Survival of bacteria and amoeba was checked at regular intervals, coupled with microscopy. It was discovered under our test conditions, that E. coli was ingested and digested by A. polyphaga, but in contrast, L. monocytogenes, had the capacity to flourish in the presence of A. polyphaga. We also report, for the first time, that all six MRSA isolates tested, survived and replicated in association with A. polyphaga, in comparison to conditions where amoebae were absent. Indeed, we also have evidence suggesting that increases in MRSA, in the presence of A. polyphaga, may be attributable to intracellular survival and replication. These findings have profound implications for the hospital environment, where Acanthamoeba sp., are also commonly isolated. In conclusion, this study illustrates the significance of protozoa as vehicles augmenting the survival of MRSA and L. monocytogenes in the environment.
Protozoa and pathogenic bacteria: lessons learned from Legionella pneumophila
Authors: SMITH A. W.
Published: April 2005

The microhabitats favoured by protozoa are dynamic ecosystems comprising a heterogeneous microflora of bacteria and fungi as well as protozoa in a complex biofilm. Many protozoa feed by grazing on microbial biofilms, but they do not do so indiscriminately. A dynamic equilibrium will exist and the nature of the relationship changes depending on environmental conditions. The view of protozoa as the hunter and bacteria as the hunted is now recognised as overly simplistic. Some bacteria not only resist predation by protozoa, but also have become endosymbionts of free‐living protozoa, demonstrating adaptation to the otherwise hostile intracellular environment. The concept that protozoa can be reservoirs for some pathogens is more widely appreciated and the catalyst for much of this work came from the observation that Legionella pneumophila infects and multiplies within some species of free‐living amoebae. In addition to amplifying cell numbers, the phenotype of intra‐protozoal‐grown cells is quite distinct from their free‐living counterparts cultured on complex laboratory media: altered surface properties, decreased susceptibility to chemical inactivation and antibiotics, and enhanced invasiveness for mammalian cells. There is significant conservation in the genes used by L. pneumophila to parasitize protozoa and macrophages and it has been proposed that co‐evolution of bacteria and lower order eukaryotic cells has equipped bacteria both for environmental survival and virulence towards higher order eukaryotes. Other authors have even described protozoa as an “evolutionary gym” enabling L. pneumophila to “train” for its role in invading higher eukaryotic organisms.

A growing list of bacteria and fungi that interact with protozoa confirm that the co‐evolution between L. pneumophila and protozoa is not unique in nature. An emerging theme amongst many of the micro‐organisms associating with protozoa is that they typically are recognised as opportunistic pathogens and many lack a recognised animal host. These features are suggestive of a relationship much closer to symbiosis which becomes parasitic only in special circumstances. The question remains why this is so? The answer could lie in co‐evolution with lower order eukaryotic organisms present in biofilms.
Study of PfMyb1 Transcription Factor Regulation Network during Plasmodium falciparum Erythrocytic Cycle
Authors: GISSOT M. , BRIQUET S. , BOSCHET C. , REFOUR P. , VAQUERO C.
Published: April 2005

During the complex life cycle of Plasmodium falciparum, the regulation of events that occur during the erythrocytic cycle, such as proliferation and differentiation, implies a fine control of transcriptional activities governing the expression profiles of each gene. However, transcriptional regulation and notably its actors, transcription factors and regulation motifs, are poorly described in Plasmodium. In order to decipher the mechanisms implicated in transcriptional regulation, we studied a transcription factor belonging to the trytophan family and showed that the PfMyb1 protein contained in nuclear extracts has a specific DNA binding activity. We took advantage of long pfmyb1 double stranded RNA (dsRNA) to inactivate the cognate messenger and understand the role of PfMyb1 during the erythrocytic cycle. Culture treated with pfmyb1 dsRNA exhibited a 40% growth inhibition and mortality during trophozoite to schizont transition when compared to either untreated control or culture treated with unrelated long dsRNA. We have further demonstrated that pfmyb1 transcript and protein decreased up to 80% in treated trophozoite culture at the time of pfmyb1 expression peak. Thus, we investigated the effect of this partial loss of transcript and protein using a thematic DNA microarray containing PCR products, representative of P. falciparum genes involved in cell cycle and transcriptional regulation. SAM software enabled us to identify several genes over and under‐expressed, potentially directly or indirectly regulated by PfMyb1. These alterations of expression were verified by qPCR and Western blotting. We are currently working on the promoters of those genes to decode determinants of gene regulation by Pfmyb1.
Protective Role of γδ T Lymphocytes during Plasmodium falciparum Infection
Authors: LOIZON S. , CONTAMIN H. , BOURREAU E. , MICHEL J.‐C. , PUIJALON O. , BEHR C.
Published: April 2005

Human T γδ lymphocytes, bearing Vγ9 Vδ2 chains, constitute a small proportion (1–5%) of the lymphocytes in the blood. These cells recognize nonpeptidic molecules such as phosphoantigens, intermediary metabolites products by microorganisms, including parasites. These antigens are recognized in the absence of molecules CMH. Our previous results have shown that Plasmodium falciparum synthetize phosphoantigen, intermediary products of the Rohmer pathway. These phosphoantigens are able to induce the proliferation of T γδ lymphocytes, producing pro‐inflammatory cytokines and providing a parasitotoxic activity against parasitized red blood cells. Also, during the acute phase of infection, these lymphocytes increase both in percentage and numbers. Today, the biological role of T γδ lymphocytes remains poorly understood. Indeed, these lymphocytes are found in primates but there is no equivalent in rodents. We decided to study this population in the infection model Plasmodium falciparum/spleen intact Saimiri scirieus. In this monkey, we found a population of TVγ9 (representing 0.2–1% of total lymphocytes in the peripheral blood) which react with phosphoantigens as do human TVγ9 cells. Activation kinetics of these cells in infected monkeys show an early activation occuring on the 6th day of infection, while the CD4 T activation starts at day 10. Also, we observed an important increase of numbers and percentages of these TVγ9. Their maximal peak coincides with the beginning of parasite clearance. Together, these data associated with parasitotoxic activity in vitro argue for a role of TVγ9 cells in parasitemia control, particulary during primary infection.
Membrane skeleton in Paramecium: a functional analysis of epiplasmins by RNAi
Authors: POMEL S. , DIOGON M. , BOUCHARD P. , BAYLE D. , RAVET V. , HENOU C. , VIGUES B.
Published: April 2005

The membrane skeleton, also called epiplasm, is a prominent cortical structure in the ciliate Paramecium. In the present study, we have developed RNAi of Ep31 and Ep34, two homologous epiplasmic proteins (epiplasmins) for a functional analysis of epiplasmic membrane skeleton in P. tetraurelia. A severe phenotype is evident 20 h after the induction of RNAi. In this phenotype, anlagen of daughter cells are formed but are unable to separate from each other at time of cytokinesis. This abortive division is repeated every 4–5 h leading to “monstrous” organisms that will die 70–75 h after induction. Immunofluorescence microscopy was performed in order to compare organization and dynamics of cytoskeletal systems in wild types and in cells processed for RNAi of epiplamins. Microtubule‐based systems are not affected as judged by examination of rows of basal bodies (bb) and bb‐associated fibers. Likewise, new oral apparatuses and pulsatile vacuoles are correctly positioned in anlagen of daughter cells. The main abnormalities concern the epiplasmic membrane skeleton. Immunolabeling of epiplasmins revealed significant disorganization of the epiplasm in regions close to aborted division furrows. Western blot analysis confirmed co‐silencing of Ep31 and Ep34 epiplasmins. These results suggest that epiplasmin‐mediated elongation of the cell body is essential for growth of the division furrow and accomplishment of binary fission in Paramecium tetraurelia.
Multigenic Family Analysis among the Encephalitozoon Genus
Authors: DIA N. , TOGUEBAYE B. S. , VIVARES C. P. , CORNILLOT E.
Published: April 2005

Encephalitozoon cuniculi is a parasite presenting a wide range of hosts among mammals (including human). Bioinformatic analysis of the genome sequence (11 chromosomes for a haploid genome size of 2.9 Mbp) revealed the presence of multigenic families in a subterminal position on the chromosomes (in 5′ of the rDNA copies on 22 extremities). One of these families was called InterB and clustered together about 30 genes within 10 homology groups. According to the predictions, the typical InterB protein presents in its N‐terminal part a signal peptide, followed by two transmembrane domains. The core of this protein is characterized by an α hypervariable region rich in glutamine and lysine. The C‐terminal region presents several conserved motifs (GGAR, YHL and FQI).

A set of specific and generic oligonucleotides was designed to study the InterB genomic repertoire. The migration profile of the generic primers PCR products presents five to six bands. This profile is well conserved for the E. cuniculi isolates as well as for the different Encephalitozoon species. Some polymorphism is evident with amplicons of 300 bp in average. Sequence analysis revealed a higher level of polymorphism, especially in the α region. The InterB repertoire of transcripts was studied by reverse transcription. All InterB genes should be transcribed. There is no known homologue for InterB but, considering the position of the genes, we speculate on the possible role of InterB proteins. Similar multigenic family organizations have been described for proteins involved in pathogenicity of parasites such as Trypanosoma, Plasmodium, Giardia or Pneumocysti.