bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Approaches to the Investigation of Viable Dinoflagellate Cysts in Natural Sediments from Estuarine Environments1
Authors: COYNE KATHRYN J. , CRAIG CARY S.
Published: April 2005

Abstract. Molecular methods offer an efficient alternative to microscopic identification of dinoflagellate cysts in natural sediments. Unfortunately, amplification of DNA also detects the presence of dead cells and is not a reliable indication of cyst viability. Because mRNA transcripts are more labile than DNA, the presence of specific transcripts may be used as a proxy for cyst viability. Here, we evaluate mRNA detection capabilities for identification of viable cysts of the dinoflagellate, Pfiesteria piscicida, in natural sediment samples. We targeted transcripts for cytochrome c oxidase subunit 1, cytochrome b (COB), and Tags 343 and 277, recently identified by serial analysis of gene expression. Expression was confirmed in laboratory cultures and compared with natural sediment samples. Three of the transcripts were detected in sediments by RT‐PCR. The fourth transcript, for COB, was not detected in sediments, perhaps because of down‐regulation of the gene in anoxic conditions. Our results suggest that methods targeting specific mRNA transcripts may be useful for detection of viable cysts in natural sediment samples. In addition, dinoflagellate cysts, which sustain extended periods of anoxia, may provide an important source of data for studies of anoxia tolerance by microbial eukaryotes.
Contrasting ciliate species richness and abundance in soils derived from Tectona grandis and Canella winterana: testing the plant effect
Authors: ACOSTA‐MERCADO DIMARIS , LYNN DENIS H.
Published: April 2005

We still have a rudimentary understanding about the mechanism by which plant roots may stimulate soil microbial interactions. A biochemical model involving plant‐derived biochemical fractions, such as exudates, has been used to explain this “rhizosphere effect” on bacteria. However, the variable response of other soil microbial groups, such as protozoa, to the rhizosphere suggests that other factors could be involved in shaping their communities. Thus, two experiments were designed to (a) obtain a better understanding of the mechanism by which ciliate species richness and abundance differ among plant species and (b) to determine whether this mechanism is maintained via stimulatory and/or inhibiting factors associated with particular plant species. Bacterial and chemical slurries were reciprocally exchanged between two plant species known to differ in terms of ciliate species richness and abundance (i.e., Canella winterana and plantation Tectona grandis). The ANOVA showed that the bacteria plus nutrients, and the nutrients‐only treatment have no significant effect on the overall ciliate species richness and abundance when compared to the control treatment. However, the use of only colpodean species to increase the taxonomic resolution of treatment effects showed that bacterial slurries have a significant effect on colpodean ciliate species richness. These results suggest that for particular rhizosphere ciliates, biological properties, such as bacterial diversity or abundance, may have a strong influence on their diversity and possibly abundance. These results are consistent with a model of soil bacteria‐mediated mutualism between plants and protozoa.
Saccamminid foraminifers from anoxic sediments: implications for Proterozoic ecosystem dynamics
Authors: BERNHARD JOAN M. , HABURA ANDREA , BOWSER SAMUEL S.
Published: April 2005

Eukaryote origin and earliest diversification occurred in the Proterozoic when Earth's atmosphere was undoubtedly different from that of today. Atmospheric oxygen levels were increasing from the primordial anoxic atmosphere due, for example, to cyanobacterial oxygenic photosynthesis. Late Proterozoic (∼0.6–0.9 Gya) deep‐ocean oxygen concentrations are less certain, but geochemical evidence suggests anoxia and hydrogen‐sulfide enrichment. It can, therefore, be postulated that initial eukaryotic diversification occurred in oxygen‐depleted, sulfide‐enriched environments. Foraminifera are aerobes and, thus, not expected in anoxic settings. Recently, however, we found a saccamminid allogromian in a deep‐water anoxic, sulfidic setting. Samples were collected from Santa Barbara Basin (California) when bottom‐water oxygen was undetectable and sediments smelled strongly of hydrogen sulfide. Foraminiferal SSU rDNA sequences recovered from sediments included one from a previously uncharacterized saccamminid. Ultrastructural analysis indicated the presence of intact Golgi, mitochondria, and prokaryotic endobionts. Saccamminid occurrence in environmental conditions known to exist during the Proterozoic supports the possibility of their origin early in eukaryotic evolution. Extant saccamminids could have competed well in the prokaryote‐dominated Proterozoic benthic ecosystem given their diet includes bacteria, bacterial biofilms and unicellular algae. Thus, Proterozoic foraminifers may have been top carnivores.

Funded by NASA NRA‐01‐01‐EXB‐057, the Geological Society of America's W. Storrs Cole Memorial Research Award, and NSF OPP0003639.
Ciliates as a tool to assess planktonic patchiness: the case of Myrionecta rubra
Authors: BULIT CELIA
Published: April 2005

Protists, and especially planktonic ciliates, exploit space by forming distinct patches at different scales. Powerful new statistical techniques for the analysis of structure in spatial and temporal distributions are being developed. One of these, geostatistics, has been used to assess patchiness of the autotrophic ciliate Myrionecta rubra in a tropical coastal lagoon. Patchiness varies with scale and over time: M. rubra forms patches of different size, and abundance exhibits a temporal pulse‐like pattern. While large‐scale patches (∼2000 m) are likely to be related to fresh‐and marine water influx and seasonal variations in nutrient inputs, mesoscale patches (100–1000 m) may originate by dissipation of larger patches, migration and growth. Fine‐scale patches (1–100 m) may be formed and advected by physical–behavioural aggregation of cells.
Paramecium aurelia revisited
Authors: COLEMAN ANNETTE W.
Published: April 2005

A major running battle was fought in the 1940s, 1950s and 1960s in the continuing war between advocates of biological species versus taxonomic (morphological) species. The subject was the taxonomic treatment of Paramecium aurelia. At that time, proponents of the newly accredited concept of a biological species (Mayr 1948) demanded that the 16 sexually isolated syngens of Paramecium aurelia be named as separate species. T. M. Sonneborn, who was largely responsible for the mating studies revealing the presence of multiple syngens (isolated mating groups) within the morphologically uniform P. aurelia, felt that species descriptions requiring biochemistry or cell culture for identification were impractical. Finally, in 1975, he capitulated, describing P. primaurelia, P. biaurelia, etc., on isozyme and mating affinity criteria. P. aurelia is not the only taxonomic species encompassing multiple cryptic species. The appropriate taxonomic solution is still moot, but we have obtained DNA sequence data now that reveal the relationships among the P. aurelia syngens, and that can be used to compare with similar cases, both in ciliates and in the microalgae, as well as plants and animals.
Short‐term dynamics and long‐term recolonization of protozoa in soil
Authors: ADL SINA M. , COLEMAN DAVID C.
Published: April 2005

Most studies of Protozoa in the soil are based on the “most probable number” (MPN) estimates from cultured sub‐samples. This approach has been criticized in recent years by protistologists. In order to work around these criticisms, we have tried to develop a set of procedures that rely on direct counts, without culturing. We show that the method is more sensitive and requires less effort than the MPN approach. Our species extractions focus on “active species at the time of sampling”, and we tried to estimate the variation between days, as species adjust to soil moisture and temperature changes over several days. These variations are compared to species composition and abundance fluctuations observed between seasons, in decomposing leaf litter bags. We also obtained abundance and community structure data based on samples from a 25‐year agro‐ecosystem chronosequence under no‐tillage management. We found that direct count methods, without prior culturing of samples, were more sensitive in detecting changes over several days, over several months, and in decadal succession from field samples. This provides a great advantage over culture‐based methods that generally fail to distinguish between encysted and re‐activated species.
The Tetrahymena thermophila genome sequence. 2. Relating macronuclear sequence scaffolds to corresponding macronuclear chromosomes
Authors: DelTORO CHRISTINA , WILLIAMSON SONDRA , ORIAS EDUARDO , HAMILTON EILEEN P.
Published: April 2005

Tetrahymena's highly fragmented macronuclear genome contains ∼300 chromosomes that have recently been whole‐genome‐shotgun sequenced at TIGR. We had genetically mapped Random Amplified Polymorphic DNAs (RAPDs) to Tetrahymena's micro‐ and macronuclear genomes. We sized the MAC chromosomes that carry RAPDs by cloning and labeling RAPD DNA and probing Southern blots of macronuclear DNA separated by pulsed‐field electrophoresis. We now report using cloned RAPD sequences to blastn‐search the TIGR Tetrahymena scaffold database (http://www.tigr.org/tdb/e2kl/tgg/); each RAPD sequence matched a sized scaffold. Comparison of physical sizes of MAC chromosomes (as determined by hybridization) to corresponding scaffold sizes showed that:

Every complete scaffold (assembled from telomere to telomere) matches in size with its corresponding MAC chromosome within 10% experimental error.

No incomplete scaffolds (telomere‐capped at one or neither end) exceeded the size of its corresponding MAC chromosome.

This work is allowing us to relate the genetic, physical and sequence maps of the micro‐and macronuclear genomes to one another. It has also provided strong evidence for the quality and accuracy of the Tetrahymena genome sequencing and assembly carried out at TIGR. (Supported by NIH grant RR02931 and contract to TIGR NIGMS Tetrahymena genome sequencing grant. Sequence data were obtained from The Institute for Genomic Research website.)
Molecular markers for assessing community diversity of coastal ciliates
Authors: DOHERTY MARY , COSTAS BARBARA , SNOEYENBOS‐WEST OONA L. O. , MCMANUS GEORGE , KATZ LAURA
Published: April 2005

Biogeography of microbial eukaryotes is widely debated. While some claim that all microbial organisms have a cosmopolitan distribution, other data suggest evidence of endemism. To assess these hypotheses for the distribution of ciliates in the orders Choreotrichia and Oligotrichia, coastal community samples were taken from Long Island Sound. We are obtaining sequences of SSU rRNA, ITS rRNA, and mitochondrial genes to use as molecular markers for assessing gene flow across time and space. These data will serve as a basis for continued study of phylogeographic distribution of Choreotrichia and Oligotrichia.
Morphological and molecular analysis of bromeliad‐inhabiting ciliates in Ecuador
Authors: DUNTHORN MICAH SAUL
Published: April 2005

The microbial inhabitants of Neotropical forests are poorly known. According to Finlay (2002, Science, 296:1061–1063) the majority of taxa in tropical forests should be previously described elsewhere. Conversely, Foissner (1999, Protist, 150:363–368) points to examples of endemic protists, and states that while we can identify the common species there are few trained taxonomists to identify the rare ones in large sampling investigations. To test these hypotheses, I am following up on an initial study of Foissner et al. (2003, Europ. J. Protistol., 39:365–372) of the ciliates inhabiting waters in canopy, sub‐canopy and terrestrial bromeliad tanks in two Ecuadorian forests. Using morphological (protargol, silver nitrate, and silver carbonate stains; scanning electron micrographs) and molecular methods (SSU and ITS sequencing) I will concentrate my investigations on the colpodid taxa found free‐living in the bromeliad tanks in two Ecuadorian forests. If I find many new taxa, Finlay's hypothesis may not be applicable to atleast bromeliad‐inhabiting ciliates.
Identification of rumen ciliates using small subunit ribosomal RNA (18S‐rRNA)‐targeted oligonucleotide probes and fluorescence in situ hybridization (FISH)
Authors: FRIED JOHANNES , WEBER SILVIA , LUDWIG WOLFGANG , McEWAN NEIL , NEWBOLD JAMIE , MICHALOWSKI TADEUSZ , THOMAS NADINE , KISIDAYOVA SVETLANA , HACKSTEIN JOHANNES , SCHLEIFER KARL HEINZ
Published: April 2005

Up to half of the biomass in the rumen can be represented by ciliates, which play an important role in the digestion processes of their hosts. In the literature, very little information can be found on determination of the diversity of complex rumen ciliate communities. The identification of these fast moving protists is mainly based on live observations and comparisons of their highly variable cell morphologies. It makes accurate identification and quantification of rumen ciliates very difficult if not impossible. The development of fluorescence in situ hybridization (FISH), established as a technique to identify prokaryotes and eukaryotes using ribosomal RNA‐targeted fluorescently labeled oligonucleotide probes, is a promising approach to identify and investigate rumen ciliates. The present study, part of CIMES (ciliates as monitors for environmental safety), a project sponsored by the European Commission, shows the problems of applying FISH on rumen ciliates and how to solve them. Tests resulted in a new protocol, which recommends para‐formaldehyde and formaldehyde at 1–2% final concentration to preserve the ciliates before applying FISH. Furthermore, seven new oligonucleotide probes could be developed and successfully be tested to identify different rumen ciliate taxa of the order Entodiniomorphida (class Litostomatea) by applying FISH. It is also shown, how FISH together with confocal laser scanning microscopy can improve analyses of ciliate cell morphologies. Thanks to Peter Pristas, Peter Javorsky, Ralf Einspanier, Susanne Ulbrich (providing rumen samples), Seung Yeo Moon‐van der Staay, Georg van der Staay (providing unpublished 18S‐rDNA sequences), the European Commission (financial support, project QLK3‐CT‐2002‐02151).
Phylogeny of lobose amoebae based on actin and small‐subunit ribosomal RNA genes
Authors: FAHRNI JOSÉ F. , BOLIVAR IGNACIO , BERNEY CÉDRIC , NASSONOVA ELENA , SMIRNOV ALEXEY , PAWLOWSKI JAN
Published: April 2005

Lobose amoebae are abundant free‐living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyse actin and small‐subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, as well as the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well‐defined clades of amoebae, the “Gymnamoebia sensu stricto” and the Archamoebae (pelobionts+entamoebids), and one weakly supported and ill‐resolved group comprising some naked, lobose amoebae and the Mycetozoa.
The Tetrahymena thermophila genome sequence: 3. Completing macronuclear chromosome sequence scaffolds and ordering and aligning them along the micronuclear genome
Authors: HAMILTON EILEEN P. , WILLIAMSON SONDRA , DelTORO CHRISTINA , ORIAS EDUARDO
Published: April 2005

In Ciliates, the diploid, mitotic, genetically silent micronucleus (MIC) is the cell's germline, while the macronucleus (MAC) is the exclusive site of gene expression. During (postzygotic) MAC differentiation in T. thermophila, several hundred chromosomes are generated by site specific fragmentation of the five germline‐derived chromosomes at highly conserved chromosome breakage sites. Telomeres are added to every newly generated end, and chromosomes are amplified to ∼45 N. The Tetrahymena thermophila macronuclear genome was recently whole‐genome‐shotgun sequenced to nine‐fold redundancy at TIGR. The sequence (publicly available at http://www.tigr.org/tdb/e2k1/ttg/) is assembled into contigs (contiguous segments of overlapping sequence reads) linked into scaffolds. Here, we report here the completion of the assembly of many scaffold ends by linking them to their corresponding telomere, using diverse bioinformatic or experimental approaches. 120 chromosomes are now assembled from telomere to telomere and 125 additional scaffolds are telomere capped at one end. The 120 complete scaffolds encompass ∼45% of the ∼105‐Mb assembly, suggesting a total of ∼290 MAC chromosomes. By using paired sequence tags flanking chromosome breakage sites, we are linking unambiguously ordered and oriented MAC chromosome DNA sequence into MIC genome assemblies.

Supported by NIH grant RR02931. Preliminary sequence data were obtained from The Institute for Genomic Research website.
Remarkable salt tolerance of naked amoebae from the Salton Sea and beyond
Authors: HAUER GWEN , ROGERSON ANDREW
Published: April 2005

This study documents the salinity tolerance of naked amoebae from the Salton Sea, California, from intertidal sand in Dania Beach, Florida, and from freshwater. Amoebae were collected from hypersaline ponds around the perimeter of the Salton Sea, where salinities averaged 160‰, and directly from the shoreline waters of the Sea where salinities were generally between 44 and 48‰. Naked amoebae were also collected from the intertidal zone of a Florida beach, a habitat subject to salinity fluctuations in the range 6–85‰. From these sites, six clones of amoebae were isolated for subsequent salinity tolerance experiments (two marine beach isolates, two Salton Sea isolates, and two hypersaline pond isolates). A seventh clone, Acanthamoeba polyphaga, a common freshwater/soil amoeba, was obtained from the Culture Collection of Algae and Protozoa (CCAP), England, UK. The culture experiments compared the effects of gradually changing salinity versus no salinity acclimatization and used growth rate and culture yield as indices of effect. Generally, amoebae were tolerant over a wide range of salinity conditions and were not markedly influenced by pre‐conditioning to salinity throughout the experiments. For example, one Salton Sea species, Platyamoeba pseudovannellida n.sp. Hauer, Rogerson & Anderson 2001, was able to survive and grow well without pre‐adaptation over the range 0–150‰. Overall, the freshwater amoeba Acanthamoeba grew between 0‰ and 12‰, the marine clones grew in the range of 0–110‰, and the Salton Sea clones reproduced between 0‰ and 150‰. The hypersaline clones were the most resilient and grew between 0‰ and 270‰. The results suggest that most amoebae are essentially unaffected in terms of growth and yield by moderate and severe salinity changes. This might partly explain the high abundances of amoebae found by the authors in salt‐challenged environments. Amoebae in the Salton Sea reached 237,120 cells/liter while those in intertidal sand ranged from 181 to 8,473 amoebae/cm3. The survival and activity of large populations of naked amoebae in sites subject to salinity fluctuations suggest that they should be considered in future studies to better understand their, as yet, undefined ecological role.
Chemosensory responses of Trichomonas to polyamines
Authors: HE INGRID , YARLETT NIGEL , LEVANDOWSKY MICHAEL
Published: April 2005

Trichomonas vaginalis, a sexually transmitted parasitic flagellate of the human urogenital tract, requires the polyamine spermine, which is present in high concentrations in the vagina and the epididymus. We investigated the possibility that these parasites might use spermine or other polyamines as chemical signals in migrating to sites of infection, which in turn would suggest a possible class of chemotherapeutic targets. We tested this possibility with a novel assay method. Log‐phase cells were placed in one of two 2‐ml tubes connected by a glass capillary; the other tube contained a test chemical. Numbers of cells migrating through the capillary into the second chamber were monitored over time by sampling the second tube. Significant migration responses were found to be 0.02 mM for spermine, putrescine, and citrulline. Lesser responses were found to spermidine, arginine and ornithine. Other amino acids did not differ from buffered salt solution controls. Cell behavior and motility were studied by videomicrosopy. A larger percentage of motile cells was observed in the presence of the active polyamines than in controls, where more cells attached to surfaces. These results suggest that this parasite does have chemosensory responses to polyamines specifically, and that the behavioral mechanism may involve a simple kinesis, though the latter possibility needs to be tested further.
Highly divergent SSU rRNA genes found in the marine ciliates Myrionecta rubra and Mesodinium pulex
Authors: JOHNSON MATTHEW D. , TENGS TORSTEIN , OLDACH DAVID W. , DELWICHE CHARLES F. , STOECKER DIANE K.
Published: April 2005

Myrionecta rubra and Mesodinium pulex are among the most commonly encountered planktonic ciliates in coastal marine and estuarine regions throughout the world. Despite their widespread distribution, both ciliates have received little attention by taxonomists. In order to understand the phylogenetic position of these ciliates better, we determined the SSU rRNA gene from cultures of M. rubra and M. pulex. Partial sequence data were also generated from isolated cells of M. rubra from Chesapeake Bay. The M. rubra and M. pulex sequences were very divergent from all other ciliates, but shared a branch with 100% bootstrap support. Both species had numerous deletions and substitutions in their SSU rRNA gene, resulting in a long branch for the clade. This made the sequences prone to spurious phylogenetic affiliations when using simple phylogenetic methods. Maximum likelihood analysis placed M. rubra and M. pulex on the basal ciliate branch, following the removal of ambiguously aligned regions. Fluorescent in situ hybridization probes were used with confocal laser scanning microscopy to confirm that these divergent sequences were both expressed in the cytoplasm and nucleolus of M. rubra and M. pulex. We found that our sequence data matched several recently discovered unidentified eukaryotes in Genbank from diverse marine habitats, all of which had apparently been misattributed to highly divergent amoeboid organisms.
Experimental studies of the relative effects of leaf litter from four hardwood trees on protozoan community productivity
Authors: ANDERSON O. ROGER
Published: April 2005

Leaf litter is an important source of organic matter as nutrients for aquatic and terrestrial microbial communities. Biogenic decomposition of leaf litter contributes substantially to atmospheric CO2, possibly producing global warming, and comprises an important part of the terrestrial carbon cycle. Small‐scale variability in terrestrial protozoan communities may be attributed to localized variations in leaf matter. This study examined the effect of aqueous leaf extracts from four trees (beech, maple, red oak, and white oak) on the abundance and diversity of protozoa in laboratory aquatic and soil cultures. Beech leaf extract was the most productive followed in the descending order by white oak, red oak, and maple in both aquatic and soil microcosm cultures. Dilution experiments indicated that concentration‐related inhibitory substance(s) in the maple and red oak leaves contribute to lower productivity. Removal of tannins by treatment with activated charcoal (Norit®) improved productivity especially for red oak leaf extracts, suggesting that tannins may contribute to the inhibition of protozoan standing stock. To determine if the lower productivity was mainly due to the inhibition of protozoan excystment, aliquots of thoroughly dried soil were inoculated into maple and red oak leaf extracts. A comparable growth of protozoa occurred in these treatments as in aliquots from moist soil indicating that inhibition of excystment is not a likely explanation for reduced productivity of the protozoan communities.
Modelling the mechanism of gregarine gliding using bead translocation
Authors: KING CONRAD , SLEEP JOHN
Published: April 2005

The gliding motility of gregarine protozoans has intrigued biologists for several decades but more recently key roles for actin and myosin have been established. Using the trophozoites/gamonts of Gregarina cell surface translocation of added 2 μm occured with similar properties to those displayed by overt gregarine gliding including motility rates of between 0.1 and 10 μm/s. Although this movement has been viewed as a smooth process, gliding (and bead translocation) is an erratic process. Thus a single 2‐μm bead translocation observed over 30 s at 0.2‐s intervals demonstrated movement for only 26% of this time (39/150). At the low Reynolds Nos operating here bead movement can only occur when force is developed at the cell surface (no consideration of inertia could apply). A laser trap was used to position a bead at the cell surface. Subsequently, the cell surface motor was able to carry the bead posteriorly out of this force trap of 50 pN. Using established figures for myosin energetics we estimate that the number of myosin heads at the interaction site to be in excess of 200. Porospora gigantea demonstrated gliding/bead translocation rates of up to 60 μm/s=four times the speed of myosin movement along the actin filaments of a muscle sarcomere!
Isolation and characterization of the “zooxanthellae” from soritid foraminifera and the giant clam Tridacna maxima
Authors: LEE JOHN J. , CEVASCO MEGAN , MÉDOR GÉRALDO
Published: April 2005

Various methodological approaches are being used to characterize Symbiodinium spp isolated from Marginopora vertebralis and Amphisorus hemprichii and Amphisorus sp. from the Indo‐pacific. While most of the cells in culture are non‐motile vegetative cells, dinospores occur in batch cultures. We find consistant variance among the isolates in the duration and time of appearance of motile forms. Nitrate (0.146 mM) supports higher populations of the isolates than NH4 (0.146 mM). Higher concentrations of NH4 inhibit growth. Although Fensome et al. (1993) characterized the Order Suessiales (and Family Symbiodiniaceae) as gymnnodiniphycideans in which the amphiesmal vesicals are arranged in 7–10 longitudinal series we were unable to detect any plates on the surfaces of our isolates in the SEM. We were unsuccessful in revealing plates on non‐motile forms by freeze fracture but they were present after treatment with various chemical agents (e.g. H2O2, HOCl−) and enzymes (cellulase, chitinase). They are not polygonal or arranged in the patterns anticipated for the Symbiodiniaceae. Polyclonal antibody studies of the Symbiodinium‐like isolates are in agreement with molecular systematic studies in the sense that there is a common relation. The foram endosymbiotic dinoflagellates share some common surface antigens with each other and with the few invertebrate isolates we have tested. Studies of the foraminiferal symbionts' plastids revealed considerable morphological diversity. We isolated a diversity of endosymbionts from the mantle of Tridacna maxima from the Red Sea and the Marshall Islands. These included Symbiodinium, Amphidinium, Tetraselmis, and an unidentified chlorophyte.
Assessment of damaged DNA and cell structure in Giardia lamblia trophozoites
Authors: LENAGHAN SCOTT , SUNDERMANN CHRISTINE
Published: April 2005

Control of protozoal pathogens in water and food destined for human use employs chemical and physical agents for inactivation of trophozoites and cysts. γ radiation, when given in certain doses, damages DNA as well as other cell components. Previously, we characterized damage in irradiated Giardia lamblia trophozoites by using Nomarski interference and scanning electron microscopy. To further examine cellular damage, irradiated trophozoites were viewed with transmission electron microscopy. Degranulation of cytoplasm at higher irradiation doses was observed, however, axonemes, adhesive discs, and peripheral vesicles appeared morphologically similar in treated as well as control cells. In order to determine damage to the DNA, the comet assay was employed. The comet assay is a single cell gel electrophoresis assay that measures the number of single and double strand breaks in DNA. This was achieved by embedment of live cells in low melting point agarose, lysis of cells, and then addition of an alkali solution to allow the DNA to unwind. After ample time for unwinding, the cells were electrophoresed, thus pulling the smaller broken strands away from the supercoiled DNA. We used Sybr GreenR, a DNA specific fluorescent dye, and a signal‐enhancing camera to visualize the DNA. Lengths of comet tails (corresponding to number of strand breaks) were measured for quantification of damage.
A preliminary report of protozoan diversity in the Joshua Tree National Park desert biome
Authors: LENNARTZ DAVID C.
Published: April 2005

Joshua Tree National Park represents an extreme environment for soil protists. The region is a mid‐latitude desert subject to temperature ranges of 0–41°C or more and annual rainfall of approximately 1.73 cm, with relatively rare flooding episodes that may deluge the area with as much as 14 cm within 1–2 h. The soil of this region has not been the subject of a systematic survey of protist diversity for at least 40 years. The goal of this study is to begin a systematic inventory of the soil protists of this region by collecting and analyzing soil samples by microscopy and other methods. Of particular interest is the soil within the rhizospheres around individual Joshua Trees (Yucca brevifolia) and creosote bushes (Larrea tridentata)—two of the most common plant genera in the region. Core samples allowed to dry for 1–2 months were subjected to the “non‐flooded Petri dish” culture method (Foissner 1987) to obtain specimens. Standard brightfield and phase contrast microscopy, selected staining methods and observation of motile behavior were used to establish identification of organisms. This preliminary study focuses on ciliate and amoeboid protists though heterotrophic flagellates were observed to be very abundant in most samples. An initial species diversity list along with representative images is presented.
Alveolates and stramenopiles in the coral reef microbenthos
Authors: LOBBAN CHRISTOPHER S.
Published: April 2005

Protists are notably absent from reef biodiversity checklists, with the exception of forams. A recent comprehensive compilation for Guam listed 303 forams, which indicate the level of biodiversity, yet there were only two other protozoans and no lists for benthic microalgae. My studies of the microbenthos began with some tube‐dwelling diatoms and colonial Pelagophyceae that form macroscopic, seaweed‐like colonies. More recently, I have become interested in two ciliates with symbiotic zooxanthellae, and in looking for these have begun to notice other protists. This presentation will display a “rogue's gallery” of microbenthos, beginning with the alveolates (ciliates—Condylostoma sp. and Euplotes spp. in particular; dinos and the symbioses), then some stramenopiles: diatoms and pelagophytes. The audience will be invited to identify the mystery ciliates and I hope the stills and videos will whet your appetite to study tropical reefs.
Ciliate diversity related to shrimps and blue crab
Authors: HONGWEI MA , OVERSTREET ROBIN M.
Published: April 2005

Observations of ciliates using the Chatton–Lwoff wet silver method, Wilbert's protargol impregnation method, and silver carbonated method revealed the infraciliature and nuclear apparatus of species collected from the blue crab (Callinectes sapidus) and brown shrimp (Farfantepenaeus aztecus) from Mississippi, white shrimp (Litopenaeus setiferus) from South Carolina, northern shrimp (Pandalus borealis) from Maine, and water associated with each. Specimens were measured and photographed under an Evolution MP 5.0 RTV digital camera system. More than 26 ciliate species, belonging to 21 families in 11 orders, were observed. Among them, five species occurred on or in the blue crab, four on the brown shrimp, two on the white shrimp, and three on the northern shrimp. Of those, only three infested more than one host species. We have seen additional symbiotic species on other specimens of the same hosts but not on those reported here. A total of 16 free‐living ciliate species occurred in the systems associated with the decapods. Among them, scuticociliates were the most important ones that could infect the decapods. One of these, Uronema marinum, also occurred in the hemolymph of the blue crab and impaired its health.

The research was supported by the USDA, CSREES Grant No. 2002‐38808‐01381 and the Blue Crab Advanced Research Consortium (BCARC), NOAA, NA17FU2841.
Mechanisms of pathogenesis of the CNS pathogen, Naegleria fowleri
Authors: MACLEAN REBECCA , MARCIANO‐CABRAL FRANCINE
Published: April 2005

Primary Amebic Meningoencephalitis is a fatal disease of the central nervous system (CNS) caused by Naegleria fowleri, an ameboflagellate found in freshwater habitats throughout the world. Pathogenic and nonpathogenic Naegleria have been isolated from the environment, but mechanisms of pathogenicity are not fully understood. Activation of complement in the CNS, induction of proinflammatory cytokines, as well as direct damage to brain tissue by the amebae may contribute to the pathogenesis of infection. We examined pathogenic N. fowleri in brain tissue from mice by scanning electron microscopy. Trophozoites with prominent food cups were abundant on lysed brain tissue. Ribonuclease protection assays of brain microglial cells cocultured with amebae revealed that pathogenic N. fowleri induced the production of proinflammatory cytokine mRNAs. Additionally, we have demonstrated that pathogenic N. fowleri activate the alternative pathway of complement but are resistant to complement‐mediated lysis. Previous studies in our laboratory have determined that an 18–20 kDa protein on the surface of N. fowleri is cross‐reactive with antibodies to mammalian CD59, a complement regulatory protein that prevents lysis of target cells. Also, cDNA encoding CD59 from human erythroleukemic cells was shown to hybridize with RNA and gDNA isolated from complement‐resistant N. fowleri but not with nonpathogenic complement‐sensitive N. gruberi in Northern and Southern blots. The amebae “CD59‐like” protein may be a virulence factor and play a role in regulating complement and also serve to protect the ameba from cytolytic molecules.
Species diversity patterns of choanoflagellates in the Pettaquamscutt River Estuary, Rhode Island, USA
Authors: MENEZES SUNSHINE , HARGRAVES PAUL E.
Published: April 2005

Choanoflagellates are a large group of heterotrophic protists that play an important role in carbon transfer through the microbial loop, as they consume bacterioplankton and have also been found to be a major food source for macrozooplankton. Although many choanoflagellate species are cosmopolitan, species diversity of the acanthoecid choanoflagellates has been shown to vary substantially between study areas around the globe. A seasonal investigation of acanthoecid species diversity was conducted along the salinity gradient in the Pettaquamscutt River Estuary (PRE), Rhode Island, USA. The taxonomy of the Acanthoecidae is based upon the morphology of a complex basket‐like lorica that surrounds the protoplast. Only one previously published study has investigated the diversity of this choanoflagellate family in the western North Atlantic, specifically in the coastal waters of Newfoundland. The Rhode Island survey found that maximum species richness occurred between July and October, and some species exhibited a salinity preference in their distributions, while others had a seasonal shift in occurrence. Acanthocorbis apoda (Leadbeater) Hara and Takahashi, 1984 occurred most consistently throughout the study (55% of samples), while Diaphanoeca grandis Ellis, 1930, Diplotheca costata Valkanov, 1970, Savillea micropora (Norris) Leadbeater, 1975, Stephanoeca cupula (Leadbeater) Thomsen 1988, and Stephanoeca supracostata Hara 1996 were each present in 36‐45% of samples. The PRE survey found a four‐fold increase in species richness of acanthoecid choanoflagellates over the Canadian study, providing further evidence of the large regional variability in choanoflagellate distributions.
The twilight of sun‐animalcules
Authors: NIKOLAEV SERGEY , BERNEY CÉDRIC , FAHRNI JOSÉ , MYLNIKOV ALEXANDER P. , PETROV NIKOLAI B. , PAWLOWSKI JAN
Published: April 2005

Molecular sampling of the taxonomic diversity of the living world is nowadays a task of paramount importance. Heliozoa represents one of the major eukaryotic taxa, which remain significantly underrepresented in molecular databases. The term Heliozoa was coined to embrace organisms with a rounded body and stiff pseudopodia. Despite evidences from ultrastructural studies, which conclusively show the polyphyly of selected heliozoan groups, contemporary morphological systems retain Heliozoa as a monophyletic taxon. From the perspective of reconstructing the true phylogeny of Eukaryota, molecular approaches to analyse relationships within this large protist group are evidently necessary. Phylogenetic analysis of our data shows that the four heliozoan taxa branch either independently or within different eukaryotic phyla. The actinophryids (Actinosphaerium, Actinophrys) appear as a lineage of stramenopiles, while the desmothoracids (Clathrulina, Hedriocystis) branch within “core Cercozoa”. The position of both groups is strongly supported in all analyses and is congruent with ultrastructure‐based taxonomic revisions. The centrohelids (Chlamydaster, Heterophrys, Pterocystis, and Raphidiophrys) do not seem to be related to any particular eukaryotic phylum, in agreement with a recent molecular study. The taxopodid Sticholonche was found to branch between Polycystinea and Acantharea, two classes of radiolarians. Results obtained in this study suggest that the heliozoan body form cannot be used as a diagnostic argument to unite Heliozoa. Instead, we discriminate between the three heliomorphic taxa of independent origin, Actinophryida, Desmothoracida and Sticholonche, and propose the novel higher rank taxon Centrohelida. The term Heliozoa should thus be used solely in historical context.
A hypothesis of genome structure in marine phytoplankton
Authors: OLIVER MATTHEW J. , FINKEL ZOE V. , SCHOFIELD OSCAR M. , FALKOWSKI PAUL G.
Published: April 2005

The genome size of marine phytoplankton is in the order of 106–1011 base pairs. Within the phytoplankton it has often been assumed that dinoflagellates have extraordinarily large genomes that significantly contribute to their nutrient requirements. We test this hypothesis by compiling cell size and genome size data from across many phytoplankton lineages, and apply a simple regression model. Our results suggest that dinoflagellates do not have anomalously large genomes, but instead scale with cell size with the same slope and intercept as many other diverse phytoplankton taxa. Based on the known correlations of genome size and genome content in diverse taxa, we model the approximate expected genome structure in unsequenced lineages of marine phytoplankton. Based on this model, we hypothesize that retrotransposons play a significant role in genome size in marine phytoplankton.
Microarrays: a tool for ecologists and protozoologists!
Authors: RUBLEE P. A. , HENRICH V. C. , MARSHALL M. M. , AMOS R. , BALSER L.
Published: April 2005

A major difficulty in microbial ecology is identifying and enumerating the members of the microbial community, both prokaryotes and eukaryotes. The advent of PCR and molecular tools offers new windows into microbial communities that do not require cultivation, thus bypassing some selective artifacts and the effort required to maintain cultures. Our goal is to develop community microarrays that have the capability to assess thousands of taxa within a single sample, including important members of the microbial community. We describe the basic approach to microarray development and preliminary results from three lakes that are providing insight into microbial communities.
Colchicine induces nuclear death in Tetrahymena thermophila
Authors: SINGHAL SITA , WOLFE JASON
Published: April 2005

We have induced the resorption of the micronucleus in living conjugants of Tetrahymena. After pairing, but prior to meiosis, the micronucleus undergoes an extraordinary elongation, expanding from a sphere of about 1‐μm diameter to a ribbon‐shaped structure whose length is greater than that of the cell. Previous EM studies showed an organized basket of microtubules subjacent to the nuclear envelope in the elongating micronucleus. We exposed early pairs to colchicines (1–5 mM), a microtubule inhibitor, and found that micronuclear elongation was prevented. Further, colchicine caused the collapse of already elongated micronuclei. Unexpectedly, with increased time of exposure to colchicine, micronuclei gradually disappeared from view; by 5 h ∼60% of cells in pairs lost their micronuclei. Direct observation ruled out ejection from the cell. We present evidence that microcnuclei are degraded by the lysosmally mediated process of autophagy. Degrading micronuclei in living pairs exposed to both Hoescht 33342 and Acridine Orange stain yellow—blue (for DNA) and orange (for acidification)—as expected for an autophagic vacuole. Further, 3‐methyladenine, an inhibitor of autophagy, blocked acidification and prevented degradation. Also, based on the requirement of actin filaments to form autophagosomes, we tested Cytochalasin B, an actin inhibitor. Cytochalasin prevented acidification and blocked micronuclear degradation. Finally, degrading micronuclei, uniquely, fluoresced red in cells whose lysosomes were pre‐loaded with Texas Red Dextran, indicate that material was transferred from lysosomes to nuclei‐containing vacuoles. These data suggest that nuclear degradation is achieved by autophagy. The mechanism by which colchicine triggers micronuclear autophagy remains to be explored.
Several mixotrophic dinoflagellates preferentially ingest ciliates as an alternate source of nutrition
Authors: SMALLEY GABRIELA W.
Published: April 2005

Mixotrophy is a common nutritional strategy among dinoflagellates. Especially in enriched coastal regions where dinoflagellates are often abundant, mixotrophy may constitute an important microbial pathway. In Chesapeake Bay, several bloom‐forming dinoflagellates are mixotrophic, including Ceratium furca, Akashiwo sanguinea, and Gymnodinium instriatum. While these three species are mainly photosynthetic, all three are capable of ingesting prey. Analysis of food vacuole content shows that they predominantly feed on oligotrich ciliates. Their grazing impact on prey populations can be substantial at times, with both C. furca and A. sanguinea capable of removing over 100% of the <20‐μm‐oligotrich standing stock. The ingested ciliate carbon, nitrogen, and phosphorus may contribute significantly to the dinoflagellates' nutritional requirements. For example, C. furca could meet up to 65% of its C, 92% of its N, and 57% of its P requirements for reproduction through phagotrophy, theoretically leading to a 40%‐reduction in doubling time. Feeding in these dinoflagellate species is influenced by a number of factors, including prey abundance, irradiance, and nutrients. In C. furca, internal carbon, nitrogen and phosphorus pools and their relative concentrations seem to play a role in feeding regulation. When cellular nutrient ratios deviate significantly from those required for optimum growth, either due to limited inorganic nutrient availability or reduced carbon fixation, feeding is induced in an attempt to balance the nutrient budget. The ability of these dinoflagellates to exploit an organic nutrient source (i.e. ciliates) may thus give them a competitive advantage over purely photosynthetic species, especially when inorganic nutrients are limited.
Molecular evolution of eukaryotes using structural genomic data
Authors: STECHMANN ALEXANDRA , CAVALIER‐SMITH THOMAS
Published: April 2005

One of the most challenging evolutionary problems is locating the root of the eukaryote tree. We know that the root lies somewhere within the kingdom Protozoa, but the widespread view that early eukaryotes were amitochondrial has recently been dramatically overturned. Systematic biases in sequence evolution prevent the reliable inference of the eukaryote root from single‐gene trees. Concatenated sequence trees should be more reliable, but leave many possibilities open. In principle deletions/insertions or gene fusions should be superior for this purpose whenever ancestral and derived states are clearly distinguishable. Using a derived gene fusion between dihydrofolate reductase (DHFR) and thymidylate synthase (TS), genes we were able to greatly narrow down the position of the root. This gene fusion clearly divides eukaryotes into two clades: Amoebozoa plus opisthokonts (unikonts, which are ancestrally uniciliate) and bikonts, which are ancestrally biciliate. Another gene fusion between the first three enzymes of the six enzyme pyrimidine synthesis pathway supports this division. This derived three gene fusion is seen in Amoebozoa and opisthokonts (unikonts) but not in bikonts. A third gene fusion in the phosphofructokinse gene which is so far only found in opisthokonts and Amoebozoa further lends support to the eukaryote unikont/bikont divide. Independent support comes from sequence trees based on concatenated protein alignments. Our results show that the primary divergence of eukaryotes was probably between unikonts (animals, Fungi, Choanozoa and Amoebozoa) and bikonts (plants, chromists, all other Protozoa: alveolates, excavates, Rhizaria, Apusozoa) and that the root of the eukaryote tree lies between these two groups.
Functional characterization of the T. gondii ortholog of parafusin by an in vivo Paramecium ortholog assay
Authors: TUCKER S. C. , LIU L. , SATIR B. H.
Published: April 2005

Parafusin (PFUS) is a phosphoglycoprotein of Paramecium that functions in Ca2+‐regulated exocytosis. The ortholog of this protein in Toxoplasma gondii, called Parafusin Related Protein 1 or PRP1, is associated with micronemes and functions in Ca2+–regulated secretion (Matthiesen et al., 2003). We have demonstrated that PRP1 colocalizes with PFUS to the Paramecium dense core vesicles (trichocysts) by means of a new assay. In this assay, recombinant, fluorescently tagged PRP1 was electroporated directly into either wild type or mutant live Paramecium cells. After a brief recovery period, the protein was observed on trichocysts docked below the cell membrane. In control experiments using labeled actin and pyruvate kinase, no localization to trichocysts was seen. Additionally, PRP1 could only be detected on undocked trichocysts that had accumulated in the cytoplasm of tam8, a Paramecium exo‐mutant that fails to transport trichocysts to the surface. Furthermore, PRP1 fluorescence disappeared from the trichocysts of wild‐type Paramecium that were stimulated with lysozyme for synchronous secretion, in a manner similar to that observed for PFUS. Cumulatively these data suggest that PRP1 of T. gondii can be properly modified, transported and localized after electroporation and that fluorochrome labeling of recombinant PRP1 did not adversely affect localization. The assay is ideal for assessing strategic residues and domains within PRP1 and other orthologs for their structural and functional relevance in exocytic processes in living cells.