Biomedical subjects

Denis H Lynn

Publications and source records attributed to Denis H Lynn.

16 recordsLinked to original sources

PubMed2006

Use of Tetrahymena thermophila to study the role of protozoa in inactivation of viruses in water.

The ability of a ciliate to inactivate bacteriophage was studied because these viruses are known to influence the size and diversity of bacterial populations, which affect nutrient cycling in natural waters and effluent quality in sewage treatment, and because ciliates are ubiquitous in aquatic environments, including sewage treatment plants. Tetrahymena thermophila was used as a representative ciliate; T4 was used as a model bacteriophage. The T4 titer was monitored on Escherichia coli B in a double-agar overlay assay. T4 and the ciliate were incubated together under different conditions and for various times, after which the mixture was centrifuged through a step gradient, producing a top layer free of ciliates. The T4 titer in this layer decreased as coincubation time increased, but no decrease was seen if phage were incubated with formalin-fixed Tetrahymena. The T4 titer associated with the pellet of living ciliates was very low, suggesting that removal of the phage by Tetrahymena inactivated T4. When Tetrahymena cells were incubated with SYBR gold-labeled phage, fluorescence was localized in structures that had the shape and position of food vacuoles. Incubation of the phage and ciliate with cytochalasin B or at 4 degrees C impaired T4 inactivation. These results suggest the active removal of T4 bacteriophage from fluid by macropinocytosis, followed by digestion in food vacuoles. Such ciliate virophagy may be a mechanism occurring in natural waters and sewage treatment, and the methods described here could be used to study the factors influencing inactivation and possibly water quality.

Animals↗

PubMed2006

Molecular phylogeny of litostome ciliates (Ciliophora, Litostomatea) with emphasis on free-living haptorian genera.

The monophyly of the litostomes was tested using nine newly sequenced and four previously unpublished small subunit ribosomal RNA (SSrRNA) gene sequences from free-living Haptoria as well as from endosymbiotic Trichostomatia: the vestibuliferids Balantidium coli and Isotricha prostoma, the cyclotrichiid Mesodinium pulex, and the haptorids Loxophyllum rostratum, Dileptus sp., Enchelyodon sp., Enchelys polynucleata, Epispathidium papilliferum (isolates A and B), Spathidium stammeri, Arcuospathidium muscorum, Arcuospathidium cultriforme, and the unusual Teuthophrys trisulca. Phylogenetic analyses depicted the litostomes as a monophyletic group consisting of the trichostomes (subclass Trichostomatia) and the free-living haptorians (subclass Haptoria). The cyclotrichiids Mesodinium and Myrionecta (order Cyclotrichiida) branched either basally within or outside the Litostomatea. In most analyses, the haptorians did not receive support as a monophyletic group. Instead, Dileptus branched basally to all litostome taxa, and Epispathidium papilliferum grouped with the Subclass Trichostomatia. Some subgroupings, however, of haptorian genera corresponded to suggested superfamilial taxa (e.g., orders Spathidiida and Pleurostomatida). Within the monophyletic trichostomes, we can distinguish three clades: (1) an Australian clade; (2) the order Entodiniomorphida; and (3) the order Vestibuliferida. However, Balantidium, currently classified in the Vestibuliferida, did not group with the other vestibuliferids, suggesting that this order may be paraphyletic.

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PubMed2006

Harvest reserves reduce extinction risk in an experimental microcosm.

Overharvesting by humans threatens a substantial fraction of endangered species. Reserves have recently received enormous attention as a means of better conserving harvested resources, despite limited empirical evidence of their efficacy. We used manipulated microcosms to test whether reserves reduce extinction risk in mobile populations of harvested Tetrahymena thermophila, a ciliate. Here we show that patterns of population distribution inside and outside reserves can be accurately predicted on the basis of simple models of diffusion coupled with logistic controls on local population growth. No extinctions occurred in eight experimental trials with reserves, whereas extinction occurred in seven of eight trials without reserves, as predicted by population viability models based on stochastic population processes. These results suggest that marine reserves may be an effective means of improving long-term viability in heavily harvested fish species.

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PubMed2005

Ophryoglena hemophaga n. sp. (Ciliophora: Ophryoglenidae): a parasite of the digestive gland of zebra mussels Dreissena polymorpha.

Ophryoglena hemophaga n. sp. is described from a freshwater Dreissena polymorpha population in the Rhine delta of the Netherlands. This is the first ophryoglenine species (order Hymenostomatida, suborder Ophryoglenina) recorded as a molluscan parasite. As is typical of ciliates in the suborder Ophryoglenina, O. hemophaga exhibits a polymorphic life history with cystment and reproduction by palintomy. Trophonts were observed within digestive gland lumina, and zebra mussel hemocytes were present in some of their digestive vacuoles. The presence of a single, longitudinal tract of multiple contractile vacuoles represents its most unique feature and distinguishes it from all other described Ophryoglena spp. The number of somatic kineties of O. hemophaga (range 100 to 124) is also distinguishing as it is one of the lowest for [corrected] an Ophryoglena sp. Other characteristics of this species include: ovoid to elongate trophonts 96 to 288 microm in length, with an elongate macronucleus 41 to 65 microm in length; tomonts 50 to 150 microm in diameter producing a clear mucous cyst envelope, whose thickness is approximately half of the tomont diameter; elongated theronts 96 to 131 microm in length which emerge after 1 to 3 cell divisions taking 36 to 48 h at 20 +/- 3 degrees C. Protomonts and theronts are, respectively, negatively and positively phototactic--characteristics that likely aid in maintenance of infection in zebra mussel populations.

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PubMed2005

Cytotoxicity of metals common in mining effluent to rainbow trout cell lines and to the ciliated protozoan, Tetrahymena thermophila.

As fish cell lines and ciliates are potential alternatives to whole animals in testing environmental samples for toxicity, two cell lines from rainbow trout, RTgill-W1 and RTL-W1, and the ciliate, Tetrahymena thermophila, were compared for their sensitivity to the toxicity of five metals that are commonly found in mining effluents: copper, cadmium, zinc, nickel, and iron. Adding copper to growth medium for either cell system failed to elicit toxicity. Therefore, metal exposures were done in simple buffers, which allowed all metals to exert toxicity, except for nickel on fish cells. Cell viability was measured successfully with alamar Blue (AB) for metabolic activity and 5'-carboxyfluoroscein diacetate acetoxymethyl ester (CFDA-AM) for membrane integrity, whereas neutral red uptake, which was quantified fluorometrically, gave confounding results with copper. Cadmium was the most toxic metal to Tetrahymena and killed the ciliate at approximately a 10-fold lower concentration than needed to kill fish cells. Fish cells were more sensitive than Tetrahymena to the cytotoxic action of zinc. Copper and iron were cytotoxic to fish cells and Tetrahymena at similar concentrations. Therefore, the relative sensitivity of fish cells and Tetrahymena to the cytotoxicity of metals depended on the metal, being similar for some but very different for others.

Animal Testing Alternatives↗

PubMed2005

Scuticociliate endosymbionts of echinoids (phylum Echinodermata): phylogenetic relationships among species in the genera Entodiscus, Plagiopyliella, Thyrophylax, and Entorhipidium (phylum Ciliophora).

Morphological research on over 50 species of ciliates recorded as endosymbionts of echinoids suggests that invasion of the echinoid microhabitat occurred on at least 4 occasions. Gene sequence data confirm the phylogenetic distinctness of spirotrichean, armophorean, plagiopylean, and oligohymenophorean endosymbionts. It is also likely that oligohymenophoreans have repeatedly invaded the gut habitat. To test this hypothesis, we sequenced small subunit rRNA (SSrRNA) genes of 6 species representing the larger scuticociliate species found in the intestine of Strongylocentrotus pallidus from the northeast Pacific Ocean: Entodiscus borealis (Entodiscidae); Plagiopyliella pacifica and Thyrophylax vorax (Thyrophylacidae); and Entorhipidium pilatum, Entorhipidium tenue, and Entorhipidium sp. (Entorhipidiidae). SsrRNA genes were amplified by PCR, and sequences obtained in both directions. In all phylogenetic analyses, the scuticociliates are well supported as a clade. Entodiscus is distinct from these other echinoid taxa and is the sister taxon to the facultatively parasitic Uronema marinum. The other 5 echinoid species always form a clade whose basal species is the free-living Parauronema longum. The greatest genetic distance among these latter 5 species is less than 1.5%. This probably explains why the Thyrophylacidae and Entorhipidiidae are paraphyletic based on the SSrRNA gene sequences.

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PubMed2004

Evaluating the toxicity of Triton X-100 to protozoan, fish, and mammalian cells using fluorescent dyes as indicators of cell viability.

Three viability assays using fluorescent dyes effectively detected a loss of viability in cultures of three mammalian cell lines (H4IIE, Caco2, and HepG-2), two fish cell lines (RTgill-W1 and RTL-W1), and a ciliated protozoan, Tetrahymena thermophila, after exposure to Triton X-100, used as a model toxicant. The dyes were Alamar Blue (AB), neutral red (NR), and propidium iodide, which respectively monitored energy metabolism, lysosomal activity, and membrane integrity. A fourth fluorescent dye, 5-carboxyfluorescein diacetate acetoxymethyl ester, was problematic. For 2-h Triton X-100 exposures, mammalian cell lines were as susceptible as piscine cell lines, whereas T. thermophila was approximately twofold less sensitive as detected with AB and NR. Despite being less sensitive, cytotoxicity tests on T. thermophila could be done in spring water, which means that unlike animal cells they could be directly exposed to most industrial effluents without osmolality adjustments. Therefore, T. thermophila could be a useful complement to animal cells as alternatives to fish in toxicity testing.

Animal Testing Alternatives↗

PubMed2003

The edaphic quantitative protargol stain: a sampling protocol for assessing soil ciliate abundance and diversity.

It has been suggested that species loss from microbial groups low in diversity that occupy trophic positions close to the base of the detrital food web could be critical for terrestrial ecosystem functioning. Among the protozoans within the soil microbial loop, ciliates are presumably the least abundant and of low diversity. However, the lack of a standardized method to quantitatively enumerate and identify them has hampered our knowledge about the magnitude of their active and potential diversity, and about the interactions in which they are involved. Thus, the Edaphic Quantitative Protargol Staining (EQPS) method is provided to simultaneously account for ciliate species richness and abundance in a quantitative and qualitative way. This direct method allows this rapid and simultaneous assessment by merging the Non-flooded Petri Dish (NFPD) method [Prog. Protistol. 2 (1987) 69] and the Quantitative Protargol Stain (QPS) method [Montagnes, D.J.S., Lynn, D.H., 1993. A quantitative protargol stain (QPS) for ciliates and other protists. In: Kemp, P.F., Sherr, B.F., Sherr, E.B., Cole, J.J. (Eds.), Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, Boca Raton, FL, pp. 229-240]. The abovementioned protocols were refined by experiments examining the spatial distribution of ciliates under natural field conditions, sampling intensity, the effect of storage, and the use of cytological preparations versus live observations. The EQPS could be useful in ecological studies since it provides both a "snapshot" of the active and effective diversity and a robust estimate of the potential diversity.

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PubMedDate not supplied

Comparing a ciliate and a fish cell line for their sensitivity to several classes of toxicants by the novel application of multiwell filter plates to Tetrahymena.

Although ciliated protozoa such as Tetrahymena have many desirable properties as toxicological test organisms, their attributes would be better realized if multiple cultures could be simultaneously exposed to toxicants, quickly washed to terminate toxicant exposure, and conveniently evaluated for changes in cellular functions. Therefore, multiwell filter plates (MWFPs), manufactured primarily for biochemical applications, were used to expose Tetrahymena thermophila to copper, Triton X-100, and gliotoxin and compared to results of exposure in microcentrifuge tubes (MCTs). For MWFP, removal of toxicant solutions and retention of Tetrahymena in wells was done by placing plates on a manifold and applying pressure with a vacuum pump. Retained cells were resuspended in the same wells and their functions assessed with the fluorescent indicator dyes, Alamar blue to measure energy metabolism, and 5'-carboxyfluorescein diacetate acetoxymethyl ester to evaluate membrane integrity. For MCTs, exposures were terminated by centrifugation, and resuspended Tetrahymena were transferred to conventional multiwell plates for viability assessment with the same fluorescent dyes. Results were measured with a fluorescent multiwell plate reader and dose-response curves were obtained successfully with both procedures. However, MWFPs were much more convenient and rapid, potentially allowing 96 cultures to be processed at a time. Exposing Tetrahymena in MWFPs also allowed the ciliate and a rainbow trout gill cell line, RTgill-W1, to be compared for their sensitivity to toxicants under similar conditions of exposure and by common viability assays. Both cell systems showed toxic responses to Triton X-100 and copper at similar concentrations, but RTgill-W1 was more sensitive to gliotoxin.

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PubMedDate not supplied

Phylogenetic position of Licnophora, Lechriopyla, and Schizocaryum, three unusual ciliates (phylum Ciliophora) endosymbiotic in echinoderms (phylum Echinodermata).

Various echinoderms are colonized by species from several classes of the Phylum Ciliophora, indicating that the echinoderm "habitat" has been invaded independently on numerous occasions throughout evolutionary history. Two "echinoderm" ciliates whose phylogenetic positions have been problematic are Licnophora macfarlandi Stevens, 1901 and Schizocaryum dogieli Poljansky and Golikova, 1957. Licnophora macfarlandi is an endosymbiont of the respiratory trees of holothuroids, and S. dogieli is found in the esophagus of echinoids. A third species, Lechriopyla mystax Lynch, 1930, is a plagiopylid ciliate found in the intestine of echinoids. Host echinoderms were collected near the Friday Harbor Laboratories, San Juan Island, WA. Specimens of S. dogieli and L. mystax were obtained from the esophagus and intestine, respectively, of the sea urchin Strongylocentrotus pallidus. Specimens of L. macfarlandi were collected from the fluid obtained from the respiratory trees of Parastichopus californicus. Using small subunit ribosomal RNA (SSrRNA) sequences of these three ciliates and a global alignment of SSrRNA sequences of other ciliates, we established the following. 1) Licnophora is a spirotrich ciliate, clearly related to the hypotrichs and stichotrichs; this is corroborated by its possession of macronuclear replication bands. 2) Lechriopyla is the sister genus to Plagiopyla and is a member of the Class Plagiopylea, which was predicted based on its cytology. 3) Schizocaryum clusters in the Class Oligohymenophorea and is most closely related to the scuticociliates; there are currently no morphological features known to relate Schizocaryum to the scuticociliates.

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PubMedDate not supplied

Soil ciliate species richness and abundance associated with the rhizosphere of different subtropical plant species.

Soil protozoa, and ciliates in particular, represent a microbial group abundant in the rhizosphere with an influential role on nutrient cycling. Under laboratory conditions, ciliates regulate the size and the composition of bacterial communities, and appear to stimulate ammonification and nitrification. In spite of their important ecological role, our understanding about the factors that control their diversity and abundance in natural forest ecosystems is still rudimentary. Plant species-specific interactions have been demonstrated between plants and soil bacteria and mycorrhizal fungi, due in part to the release of phytohormones and C- and N-rich exudates. We tested the hypothesis that the rhizosphere environments of different plant species also influence the species richness and abundance of soil ciliates. Plant effect, soil pH, moisture content, microbial biomass C, and inorganic nitrogen were measured among five plant species to determine the best predictor variables for soil ciliate species richness and total abundance in a subtropical moist forest in Puerto Rico. Based on an analysis of variance, we rejected the hypothesis that there was a plant species-specific effect on soil ciliates, unlike other microbial groups mentioned above. Using multiple regression analysis, we demonstrated that the flush of total inorganic nitrogen was the best predictor variable for both species richness and abundance of ciliates.

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PubMedDate not supplied

Phylogenetic position of species in the genera Anoplophrya, Plagiotoma, and Nyctotheroides (Phylum Ciliophora), endosymbiotic ciliates of annelids and anurans.

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

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PubMedDate not supplied

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists.

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.

Animal Population Groups↗

PubMedDate not supplied

Parastrombidinopsis shimi n. gen., n. sp. (Ciliophora: Choreotrichia) from the coastal waters of Korea: morphology and small subunit ribosomal DNA sequence.

The planktonic ciliate Parastrombidinopsis shimi n. gen., n. sp. is described from both living cells and quantitative protargol-stained (QPS) preparations and the sequence of the small subunit rDNA (SSU rDNA) is reported. This species is almost oval when the cells are alive; when stained, it is cylindrical for the upper two-fifths, half-bowl shaped for the middle two-fifths, and narrow rodshaped for the lower one-fifth. The ranges (and mean +/- standard deviation, n = 20) of cell length, cell width, and oral diameter of living cells were 112-221 microm (168 +/- 39), 88-176 microm (121 +/- 30), and 53-110 microm (80 +/- 14), respectively, while those of the QPS-stained specimens (n = 54) were 88-225 microm (162 +/- 29), 55-163 microm (102 +/- 19), and 53-98 microm (69 +/- 9), respectively. Thirty-six to 48 external oral polykinetids had cilia 25-40 microm long. However, unlike Strombidinopsis species sensu stricto, P. shimi has an external oral polykinetid zone that is an open circle. This species has two shorter polykinetids associated with the end of the oral polykinetid zone, deep in the oral cavity. Like Strombidinopsis species in the subclass Choreotrichia, 36-50 somatic kineties were equally spaced around the cell body and extended from the oral to the posterior regions with 68-105 dikinetids per kinety. Both kinetosomes of each kinetid bore cilia 3-10 microm long. Parastrombidinopsis shimi had 2 (1-4) ovoid macronuclei of 20-82 x 15-32 microm. When properly aligned, the sequence of the SSU rDNA of P. shimi (GenBank Accession No. AJ786648) was approximately 5% different from that of Strobilidium caudatum and 6% different from that of two Strombidinopsis species. Based both on morphology and gene sequence divergence, we establish this is as a new species in a new genus belonging to the family Strombidinopsidae.

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PubMedDate not supplied

Species of tetrahymena identical by small subunit rRNA gene sequences are discriminated by mitochondrial cytochrome c oxidase I gene sequences.

The mitochondrial cytochrome c oxidase 1 (CO1) genes of two isolates of each of the seven mating types of Tetrahymena thermophila were sequenced and found to differ by < 1% in nucleotide sequence and to be identical by putative protein sequence. As this gene was highly conserved in this species, the CO1 gene sequence was determined for four pairs of Tetrahymena species identical in their small subunit rRNA gene sequences. The following pairs of species showed from 1% to 12% divergence at the nucleotide level, enabling discrimination of all these species: (1) Tetrahymena pyriformis strain T and Tetrahymena setosa strain HZ-1; (2) Tetrahymena canadensis strain UM1215 and Tetrahymena rostrata strain ID-3; (3) Tetrahymena pigmentosa strain UM1285 and Tetrahymena hyperangularis strain EN112; and (4) Tetrahymena tropicalis strain TC-105 and Tetrahymena mobilis. However, because of the synonymous nature of the majority of substitutions, the pairs of species were identical based on the putative protein sequence.

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PubMedDate not supplied

A java applet for exploring the new higher level classification of eukaryotes with emphasis on the taxonomy of protists.

We have converted the hierarchically organized new higher level classification of eukaryotes with emphasis on the taxonomy of protists proposed by Adl et al. into an interactive and dynamic Java applet. The current version of the applet can be accessed via http://phylogenetics.bioapps.biozentrum.uni-wuerzburg.de/etv. We use the layout from a Degree-of-Interest tree (DOITree) that effectively displays all the taxonomic information as well as the phylogenetic relationships described in the original article by Adl et al. The tree was made using the Prefuse Toolkit for interactive information visualization. All browsers capable of using Java applets will be able to view the tree. The applet is freely available for scientists, teachers, and students.

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