An Experimental Study on the Food Chain Among Bacteria,Paramecium and Daphnia

A quantitative study is made of the feeding of Paramecium caudatum on bacteria and of the feeding of Daphnia pulex on Paramecium and bacteria. The growth yield of P. caudatum on a diet of an unidentified yellow-colored bacterium was about 55% on a dry weight basis. Paramecium would not grow if the concentration of bacteria is less than 1 . 10₇ cells ml. Several other bacterial species were tested. Paramecium could not grow successfully on all of them, but could on Rhodopseudomonas spheroides. Adults of Daphnia pulex could grow and reproduce on a diet of P. caudatum alone, but young grew successfully only when bacteria were present.     

Economic Mass Cultivation of Paramecium tetraurelia on a 200-Liter Scale1

ABSTRACT. A new and inexpensive medium is described for axenic mass cultivation of Paramecium tetraurelia stock 51s and the double mutant pawn A/pawn B. Skim milk powder is the major carbon and nitrogen source in this medium. Growth characteristics (proliferation rate, final cell density, and cell size) are similar to those observed with other axenic culture media. Cultures were run in one-liter erlenmeyer flasks, a 20-liter, and a 250-liter airlift bioreactor. The yield of a large bioreactor is 750 g (wet wt.) Paramecium or 5 × 10⁹ cells. This easy, economical culture technique will greatly facilitate the use of Paramecium as a model organism for extensive biochemical studies.     

The Purification of DNA from the Genomes of Paramecium aurelia and Tetrahymena pyriformis1

SYNOPSIS. Methods are described for the recovery of highly purified DNA from Paramecium aurelia and Tetrahymena pyriformis in high yields. Our DNA is only slightly contaminated with RNA and carbohydrate, and little or no protein can be detected. We could not reduce the RNA (orcinol-positive material) by further treatment (sephadex or hydroxyapatite chromatography and preparative CsCl gradients). At the extreme our DNA is contaminated with 15–20% RNA but the real value is most likely considerably lower than this. The DNA we have prepared from Paramecium and Tetrahymena shows all the properties of double-stranded, high molecular weight DNA when characterized by temperature melting, CsCl density gradient centrifugation and hydroxyapatite and sephadex chromatography. When denatured, it absorbs to nitrocellulose filters. The 2 major results of importance from our work reported here are: (1) There is similarity in base composition of DNA from different syngens of Paramecium (28% G+C for syngens 1, 2, 4, 5, and 9 and 29–30% G+C for syngen 8) while there is variation between the syngens of Tetrahymena (24–31% G+C for syngens 1, 4, 7, 10, 11, and 12); (2) the density of any Paramecium DNA varies depending upon whether the cells are grown in the presence of bacteria or in axenic medium. Our results are compatible with observations previously reported for Tetrahymena but contradict those made for Paramecium. The earlier reports of differences in base composition between syngens of Paramecium are probably due in large part to the use of stocks grown on bacteria.     

Method for the Simultaneous Establishment of Many Axenic Cultures of Paramecium*†

SYNOPSIS. A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmasterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing ～ 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliates can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock each of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

Induction of Antibiotic Resistance in Paramecium tetraurelia by the Bacterial Gene APH-3'-II

We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neo^r) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neo^r ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10–20 picoliters of linearized PXV-NEO at > 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neo^r-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neo^r-specific DNA and neo^r-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.     

Effect of Acetate on Esterase C Activity During the Growth Cycle of Paramecium*

SYNOPSIS Enhanced esterase C activity could be demonstrated by starch gel electrophoresis in various stocks of Paramecium spp. (P. primaurelia stocks 90 and 540, P. biaurelia stock 93, P. tetraurelia stock 29. P. pentaurelia stock 87, P. octaurelia stocks 31 and 300, and P. multimicronucleatum species 3, stock 8 MO) grown in Adaptation Medium. This esterase, however, was barely detectable when they were cultivated in Axenic Medium. Addition of trypticase to Adaptation Medium resulted in reduction of esterase C in the ciliates. This effect is ascribable to Na acetate present in trypticase. Since esterase C increased with the decrease in acetate concentration (as estimated by gas-liquid chromatography) during growth of Paramecium, acetate appears to be utilized by the cells. Sensitivity of esterase C to acetate occurs in all 6 species of Paramecium examined. Different stocks within a species may have different levels of sensitivity; in one case this is genetically determined. The results emphasize the importance of controlling and manipulating growth conditions for the assessment of inter- and intraspecies variations in the isozymes of Paramecium.     

Vertically transmitted symbiont reduces host fitness along temperature gradient

Parasites with exclusive vertical transmission from host parent to offspring are an evolutionary puzzle. With parasite fitness entirely linked to host reproduction, any fitness cost for infected hosts risks their selective elimination. Environmental conditions likely influence parasite impact and thereby the success of purely vertical transmission strategies. We tested for temperature‐dependent virulence of Caedibacter taeniospiralis, a vertically transmitted bacterial symbiont of the protozoan Paramecium tetraurelia. We compared growth of infected and cured host populations at five temperatures (16–32 °C). Infection reduced host density at all temperatures, with a peak of ?30% at 28 °C. These patterns were largely consistent across five infected Paramecium strains. Similar to Wolbachia symbionts, C. taeniospiralis may compensate fitness costs by conferring to the host a ‘killer trait’, targeting uninfected competitors. Considerable loss of infection at 32 °C suggests that killer efficacy is not universal and that limited heat tolerance restricts the conditions for persistence of C. taeniospiralis.     

A Functional Aqp1 Gene Product Localizes on The Contractile Vacuole Complex in Paramecium multimicronucleatum

In a ciliate Paramecium, the presence of water channels on the membrane of contractile vacuole has long been predicted by both morphological and physiological data, however, to date either the biochemical or the molecular biological data have not been provided. In the present study, to examine the presence of aquaporin in Paramecium, we carried out RT-PCR with degenerated primers designed based on the ParameciumDB, and an aquaporin cDNA (aquaporin 1, aqp1) with a full-length ORF encoding 251 amino acids was obtained from Paramecium multimicronucleatum by using RACE. The deduced amino acid sequence of AQP1 had NPA-NPG motifs, and the prediction of protein secondary structure by CNR5000 and hydropathy plot showed the presence of six putative transmembrane domains and five connecting loops. Phylogenetic analysis results showed that the amino acid sequence of AQP1 was close to that of the Super-aquaporin group. The AQP1-GFP fusion protein clearly demonstrated the subcellular localization of AQP1 on the contractile vacuole complex, except for the decorated spongiome membrane. The functional analyses of aqp1 were done by RNA interference-based gene silencing, using an established feeding method. The aqp1 was found to be crucial for the total fluid output of the cell, the function of contractile vacuole membranes.     

Genetic diversity of Paramecium species on the basis of multiple loci analysis and ITS secondary structure models

Among ciliates, Paramecium has become a privileged model for the study of “species problem” particularly in the case of the “Paramecium aurelia complex” that has been intensely investigated. Despite extensive studies, the taxonomy of Paramecium is still challenging. The major problem is an uneven sampling of Paramecium with relatively few representatives of each species. To investigate species from the less discovered region (Pakistan), 10 isolates of Paramecium species including a standing-alone FT8 strain previously isolated by some of us were subjected to molecular characterization. Fragments of 18S recombinant DNA (rDNA), ITS1-5.8S-ITS2-5′LSU rDNA, cytochrome c oxidase subunit II, and hsp70 genes were used as molecular markers for phylogenetic analysis of particular isolates. The nucleotide sequences of polymerase chain reaction products of all markers were compared with the available sequences of relevant markers of other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all the nine strains had a very close relationship with Paramecium primaurelia except for the FT8 strain. FT8 consistently showed its unique position in comparison to all other species in the phylogenetic trees. Available sequences of internal transcribed spacer 1 (ITS1) and ITS2 and some other ciliate sequences from GenBank were used for the construction of secondary models. Two highly conserved helices supported by compensatory base changes among all ciliates of ITS2 secondary structures were found similar to other eukaryotes. Therefore, the most conserved 120 to 180 base pairs regions were identified for their comparative studies. We found that out of the three helices in ITS1 structure, helix B was more conserved in Paramecium species. Despite various substitutions in the primary sequence, it was observed that secondary structures of ITS1 and ITS2 could be helpful in interpreting the phylogenetic relationships both at species as well as at generic level.     

A Note on the Possible Ecological Significance of Chemotaxis in Certain Ciliated Protozoa1

ABSTRACT. A heat-stable chemoattractant has been isolated from bacterial cultures. This component has a molecular weight in the range of 500–1000 daltons, is produced by both Gram-positive and Gram-negative bacteria, and serves equally well as an attractant for both the bacterial feeding Paramecium and for its natural predator, Didinium. Aspects of the ecological relationship between bacterial feeding ciliates and their ciliate predators are briefly discussed with respect to responses of both predator and prey to such a common chemotactic bacterial factor.     

Population growth,demography and competition studies on Dipleuchlanis propatula (Gosse, 1886) (Rotifera: Euchlanidae)

We tested the effect of different food (Chlorella vulgaris) concentrations on the population growth and demographic variables of Dipleuchlanis propatula. In addition, we also tested the effect of competition from two other zooplankton species (a rotifer Plationus patulus and a ciliate Paramecium sp.). The parthenogenetic eggs of D. propatula carry a gelatinous matrix with a thickness of about 20 µm. Population growth of D. propatula increased with increase in the algal density. Depending on the food availability, the peak abundances of D. propatula varied from 9 to 50 ind. ml^?1. The rate of population increase, r, varied from 0.18 to 0.23 per day. Food density had a significant effect (p?<?0.05, Tukey test) on this variable too. In spite of higher peak densities observed at high food levels, the r values were not very different; this was due to the long lag phase before the population increased. Dipleuchlanis propatula growth was lower due to competition from ciliates. Food density had no significant effect on the average life span, which varied from 5 to 7 days. However, both gross and net reproductive rates increased with increase in algal food. These results show that D. propatula had growth patterns similar to other members of the family Euchlanidae.

     

Monoclonal Antibody to a Bacterial Endonuclear Symbiont Holospora Cross Reacts with Proteins of Contractile Vacuole Radial Canals of Paramecium Species

ABSTRACT A monoclonal antibody (mAb) IR-2-1 was raised against a 67-kDa protein purified from the macronucleus-specific bacterial symbiont Holospora obtusa of Paramecium caudatum. The mAb was found to react with two bands (31 and 67-kDa) on gels of H. obtusa. Indirect immunofluorescence microscopy showed that these antigens were distributed inside the cells. However, unexpectedly, this mAb also cross reacted with the radial arms of the contractile vacuole in P. caudatum, P. tetraurelia, P. multimicronucleatum, P. jenningsi and P. bursaria as well as with their cytoplasm. Immunoelectron microscopy showed that the antigens were located on the decorated spongiome of the radial arms. In immunoblots, mAb IR-2-1 reacted with a band of 67 kDa in all Paramecium species examined. However, no band appeared in the immunoblot of isolated macronuclei of H. obtusa-free P. caudatum and no label was seen in the nuclear matrix of the macronucleus of air-dried P. caudatum. These results suggest that the 67-kDa antigen found in H. obtusa was not imported from the host macronucleus and the same antigen in the host contractile vacuoles and cytoplasm were not derived from the symbiont. These results also showed that an epitope on the decorated spongiome of the Paramecium species is shared by its bacterial symbiont. In contrast to the decorated tubule-specific mAb, DS-1, the antigens for IR-2-1 appeared to be loosely membrane bound as they were lost in paraformaldehyde fixed and acetone permeabilized Paramecium. Supplementary key words. Contractile vacuole complexes, Holospora obtusa, monoclonal antibody, Paramecium.     

Biochemical Taxonomy of Symbiotic Chlorella Strains from Paramecium and Acanthocystis*

Biochemical and physiological characters of 5 symbiotic Chlorella strains, 4 of them from Paramecium (Ciliata) and one from Acanthocystis (Rhizopoda, Heliozoa), were studied. Four strains (3 from Paramecium and one from Acanthocystis) belong to the same species. This is characterized by the presence of hydrogenase, no formation of secondary carotenoids, no growth on mannitol, requirements for thiamine and vitamin B₁₂, and a G + C content of the DNA of 66.4–68.4 mol%; the limits of growth are at pH 5.5, at up to 1% NaCl, and at 26–30°C. The strains are somewhat heterogeneous in their utilization of inorganic nitrogen sources: only two of them are able to use nitrate, whereas all can grow with nitrite and ammonium. Thus, in two strains the nitrate-reducing system — in contrast to nitrite reductase — seems to be defective. Another strain, which has been claimed to be from Paramecium, belongs to C. protothecoides.     

Simultaneous recording of cytosolic Ca2+ levels inDidinium andParamecium during aDidinium attack onParamecium

Summary The carnivorous ciliateDidinium nasutum captures prey such asParamecium by discharging extrusomes, known as toxicysts, while the attackedParamecium defensively discharges trichocysts. Several authors have suggested that both discharges, the toxicysts ofDidinium and the trichocysts ofParamecium, are evoked by the rise in cytosolic Ca²⁺ level in each cell. However, these putative increases in cytosolic Ca²⁺ levels have not as yet been recorded simultaneously in these cells during aDidinium attack onParamecium. We injected the fluorescent Ca²⁺ indicator Ca-Green 1 dextran into bothDidinium andParamecium, and simultaneously observed the cytosolic Ca²⁺ levels in these cells asDidinium attackedParamecium. When aParamecium came into contact with theDidinium proboscis, theDidinium showed a significant rise in cytosolic Ca²⁺ in the basal portion of the proboscis. One video frame (33 ms) after the onset of the Ca²⁺ rise inDidinium, theParamecium also showed an increase in cytosolic Ca²⁺. This is the first simultaneous recording of changes in the Ca²⁺ level during a predator-prey interaction in ciliates. The possible roles of these Ca²⁺ increases are discussed in relation to the discharge of toxicysts during theDidinium attack and of trichocysts as a defensive behavior ofParamecium.Abbreviations AED aminoethyldextran - P_i inorganic phosphate - FITC fluorescein isothiocyanate     

Chemorepellents in Paramecium and Tetrahymena

Although Paramecium has been widely used as a model sensory cell to study the cellular responses to thermal, mechanical and chemoattractant stimuli, little is known about their responses to chemorepellents. We have used a convenient capillary tube repellent bioassay to describe 4 different compounds that are chemorepellents for Paramecium and compared their response with those of Tetrahymena. The classical Paramecium t-maze chemokinesis test was also used to verify that this is a reliable chemorepellent assay. The first two compounds, GTP and the oxidant NBT, are known to be depolarizing chemorepellents in Paramecium but this is the first report of them as repellents in Tetrahymena. The second two compounds, the secretagogue alcian blue and the dye cibacron blue, have not previously been described as chemorepellents in either of these ciliates. Two other compounds, the secretagogue AED and the oxidant cytochrome c, were found to be repellents to Paramecium but not to Tetrahymena. The repellent nature of each of these compounds is not related to toxicity because cells are completely viable in all of them. More importantly, all of these repellents are effective at micromolar to nanomolar concentrations, providing an opportunity to use them as excitatory ligands in future works concerning their membrane receptors and possible receptor operated ion channels.     

Downward regulation of macronuclear DNA content in Paramecium tetraurelia : Effects of excess DNA on the subsequent DNA and protein content and the cell growth rate

Paramecium cells were selected which received the entire parental macronucleus at fission and thus started the cell cycle with twice the normal post-fission DNA content. During each of the subsequent two cell cycles the cells synthesized approximately as much DNA as did control cells. The amount of excess macronuclear DNA was consequently halved during each cell cycle. The minimum pre-fission DNA content was just larger than the mean post-replication DNA amount, confirming that a similar amount of DNA, approximately equal to the mean post-fission DNA content of the non-selected population, was synthesized in macronuclei, regardless of the post-fission DNA content. These observations confirm a model for DNA content regulation previously devised for Paramecium and are inconsistent with DNA content regulation schemes proposed for other ciliates. The increased DNA content has no effect either on the subsequent total protein content of pre-fission cells, or on the rate of cell growth. This suggests that the rate of cell growth is limited by the size of the cell when the macronuclear gene-dosage is equal to or greater than that in normal cells. The results also suggest that the amount of DNA synthesized within an interfission period is also limited by the size of the cell and is proportional to the cell mass. Paramecium does not require a fixed nucleocy oplasmic ratio as a pre-condition either for cell division, or, by inference, for initiation of DNA synthesis.     

A New Method For Axenic Mass Cultivation of Paramecium Tetraurelia*

SYNOPSIS A method is described for the axenic mass cultivation of Paramecium tetraurelia strains 51s and 299s. the ciliate is grown in an enriched axenic medium developed by Soldo, Godoy & van Wagtendonk. Under continuous shaking on a rotary shaker, cultures were grown in one-liter Erlenmeyer flasks with 330 ml medium yield cell densities of 32,000 cell/ml and 20,000 cells/ml for strains 299s and 51s respectively. Doubling time is considerably shorter under these conditions than in the conventional static cultures. A 20-liter airlift bioreactor is described in detail which can be used successfully to otain up to 100 g wet weight of Paramecium in a single run; in this reactor the cell density reaches 38,000 cells/ml for strain 299s. and 23,000 cells/ml for 51s. This technic should facilitate the study of minor protein components of the ciliate.     

The Role of Trichocyst Discharge and Backward Swimming in Escaping Behavior of Paramecium from Dileptus margaritifer1

Trichocyst discharge is an effective defense of Paramecium against Dileptus margaritifer. The possible defensive function of backward swimming, which often follows trichocyst discharge upon Paramecium-Dileptus encounters was studied. Mutants incapable of backward swimming (pawnA in P. tetraurelia, cnrA in P. caudatum) escaped from dilepti nearly as frequently as wild-type cells. Double mutants (pawnA-nd7, cnrA-tnd2) were eaten nearly as frequently as mutants incapable of trichocyst discharge. Thus, in the defense of Paramecium against D. margaritifer, the role of backward swimming is minor, if any, compared to trichocyst discharge. Among escaped cells, about a half of wild-type and essentially none of pawnA (cnrA) cells showed backward swimming. Paramecium behavior during the encounter can be mimicked by the local, not global, application of lysozyme which is a strong secretagogue of trichocyst.     

Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium

Summary— Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the α N-terminal domain, one in the β C-terminal one, and two in α and β central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of α-tubulin whereas the epitopes of three other ones (TAP 952°, AXO 58 and AXO 49°) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both α- and β-tubulins. The AXO 49° specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.     

Investigations on influencing plant-associated bacteria in tissue cultures of black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.)

During tissue culture of black locust (Robinia pseudoacacia L.), serious problems with plant-associated bacteria led to a reduction of propagation potential in several clones. Four dominant strains of plant-associated bacteria could be isolated and were assigned to the genera Acidovorax, Dyella, Microbacterium and Sphingomonas. Out of five essential oils tested, thyme and lemongrass oil at a concentration of 0.03% each and 0.015% of both oils in combination clearly inhibited the growth of these bacteria strains on bacteriologic medium. There were no significant differences in total bacterial population density when penicillin, thyme and lemongrass oil or thyme plus lemongrass oil were added to the plant propagation media. The use of lemongrass oil changed the proportion of dominant bacterial strains.