The accidental protozoologist--my journey through the world of ciliates

The Past-President's Address has been the opportunity for the speaker to reminisce about the road traveled to get to this time in life. In this paper, I continue in that tradition. During my journey to the present day, I visited different laboratories, studying the genetics of mating and mating types in Paramecium, Tetrahymena, Blepharisma and Euplotes. I have met and worked with many distinguished scientists, including other Past-Presidents of the Society. I also became an active participant in the Society of Protozoologists. I hope the recounting of my trip will be both entertaining and enlightening.     

Automated image analysis to improve bead ingestion toxicity test counts in the protozoan Tetrahymena pyriformis

AIMS: To improve bead ingestion counts in Tetrahymena pyriformis by automated image analysis as an alternative to direct-counts. METHODS AND RESULTS: Fluorescent latex beads were added to T. pyriformis cultures for ingestion tests. The number of beads ingested by 25 cells was counted directly by epifluorescence microscopy and compared with similar data from image analysis. anova indicated that counts were not significantly different (P < 0.05). The image analysis particularly provided advantages in terms of speed. CONCLUSIONS: The image analysis is superior to direct beads counting in T. pyriformis particularly in terms of speed of analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The image analysis method is very rapid and will allow many more toxicological analyses to be undertaken with less operator error.     

Industrial media and fermentation processes for improved growth and protease production by Tetrahymena thermophila BIII

Tetrahymena thermophila was cultivated on industrial by-product media. The composition of the best medium (with milk proteins) was optimised by a central composite design for growth and protease secretion. The optimal combination [1.07% (w/v) of yeast extract and 0.99% (w/v) of skimmed milk] improved biomass production by 46%. In a fermentation strategy, the pH must be regulated to produce no cell damage, lengthening the stationary phase and resulting in a more abundant protease production. To increase cell concentration and protease secretion, a continuous culture with cell recycling by microfiltration was successfully tested on ciliated protozoa. Journal of Industrial Microbiology & Biotechnology (2000) 24, 285–290. Received 28 July 1999/ Accepted in revised form 20 January 2000     

Effect of quinacrine treatment on the activity of acid hydrolases (phosphatase, N-acetyl-beta-glucosaminidase, glucosidase, galactosidase and esterase) in Tetrahymena.

The effect of phospholipase A2 (PLA2) inhibitor, quinacrine, on the activity of hydrolytic enzymes in Tetrahymena pyriformis homogenate, was investigated. The activity of all of the enzymes studied (acid phosphatase, N-acetyl-beta-glusosaminidase, glucosidase, galactosidase and esterase) was significantly reduced in the presence of quinacrine. Since there are no data on the inhibitory effect of PLA2 and PLA2 influenced metabolic pathways to the hydrolytic enzymes, the direct effect of quinacrine on the hydrolytic enzymes (of Tetrahymena) can be supposed. This is supported by the fact that the other PLA2 inhibitor, 4-bromophenacyl bromide, did not influence phosphatase activity.     

How does the unicellular <Emphasis Type="Italic">Tetrahymena</Emphasis> utilise the hormones that it produces? Paying a visit to the realm of atto-and zeptomolar concentrations

Changes in the hormone content of Tetrahymena pyriformis GL were investigated during histamine, serotonin or insulin treatment at concentrations of 10⁻⁶M to 10⁻²¹M for 30 min. The immunologically demonstrable hormone content was studied by using specific antibodies, flow cytometry and confocal microscopy. Histamine at the higher ranges elevated the serotonin content of Tetrahymena, whereas serotonin at the lower ranges (down to 10⁻²¹M) decreased its histamine levels. Insulin did not affect its serotonin content, whereas serotonin increased its insulin content at each concentration studied (down to 10⁻²¹M). Insulin between 10⁻⁶M and 10⁻²¹M increased the hista-mine levels of Tetrahymena, although histamine influenced its insulin level only at 10⁻⁶M. Our results call attention to the presence of hormonal interactions even at “low” levels of phylogeny and to the extreme sensitivity of the hormone receptors of Tetrahymena. These data might explain (1) the requirement of Tetrahymena for (vertebrate) hormone production and hormone receptors and (2) the way that it uses these hormones under natural conditions.This work was supported by the National Research Fund (OTKA-T-037303), Hungary.     

Physiological, morphological and metabolic changes in Tetrahymena pyriformis for the in vivo cytotoxicity assessment of metallic pollution: Impact on d-β-hydroxybutyrate dehydrogenase

The individual cytotoxicity of cadmium chloride, iron sulphate and chromium nitrate has been investigated by using the freshwater ciliate Tetrahymena pyriformis. The metabolic enzymes and antioxidant defense biomarkers were assessed. The results obtained reveal that their metal salts have perturbed the physiology and morphology of T. pyriformis. Also, the biomarkers assessed were sensitive to the presence of metal salts and this sensitivity was metal salt and dose dependant. To estimate the impact of their metal salts on mitochondria, we studied their effects in vivo and in vitro on the d-β-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30) inner mitochondrial membrane enzyme. The results showed a high inhibition of BDH in terms of activity, protein expression and kinetic parameters.     

Chemoattraction to lysophosphatidic acid does not require a change in membrane potential in Tetrahymena thermophila

LPA (lysophosphatidic acid), a known chemoattractant for many types of eukaryotic cells, is also a reliable chemoattractant for Tetrahymena. Since LPA receptors are GPCRs (G-protein coupled receptors) in many cell types and several putative GPCR sequences can be found in the Tetrahymena Genome Database, we are interested to determine whether similar GPCR pathways can be used for chemosensory transduction in Tetrahymena. To confirm our procedures, we tested the known chemoattractant proteose peptone (at 1.0 mg/ml), which caused hyperpolarization and increased forward swimming speed in Tetrahymena, consistent with the current model for ciliate chemoattraction. Although 10 μM LPA did not produce these same responses, it was still an effective chemoattractant. PTX (pertussis toxin) blocked attraction to both of these compounds, suggesting a possible G-protein involvement in chemoattraction. Both of these chemoattractants also decreased the basal percent of cells showing direction changes [PDC (percent directional change)] and the duration of backward swimming in 0.5 mM Ba2+ (a general excitability assay). LPA probably causes chemoattraction in Tetrahymena by decreasing the basal PDC without changing either membrane potential or swim speed. Since a pertussis-sensitive G-protein might modulate the ciliate voltage-dependent Ca2+ channels, we propose that LPA acts through an uncharacterized GPCR to lower the PDC by decreasing cellular excitability. These combined behavioural and electrophysiological analyses support the novel hypothesis that chemoattraction to some attractants, like LPA, can occur without hyperpolarization and increased swim speed in Tetrahymena.     

Presence of a Bacterial-Like Citrate Synthase Gene in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>: Recent Lateral Gene Transfers (LGT) or Multiple Gene Losses Subsequent to a Single Ancient LGT?

Citrate synthase is the initial enzyme in the tricarboxylic acid cycle of mitochondria. In plants and fungi, it is the second isozyme in the glyoxylate cycle of peroxisomes (or glyoxysomes), and it is also present in bacteria. Some of the biochemical reactions in the glyoxylate cycle of the ciliated protozoan Tetrahymena pyriformis depend upon mitochondrial enzymes, as T. pyriformis lacks some glyoxysome-specific enzymes. Here we demonstrate a new citrate synthase gene from Tetrahymena thermophila that is different from the mitochondrial counterpart. A potential peroxysome-targeted signal was detected in the N-terminus, suggesting the localization of the enzyme in peroxysomes. Phylogenetic analysis placed the Tetrahymena sequence in a clade consisting of a few sequences from eukaryotes such as cellular slime molds and two land plants, near a green sulfur bacterium and many proteobacteria as a sister group but not in a mitochondrial clade. Southern blot analysis revealed that this type of gene was absent from distantly related ciliates and other species of Tetrahymena except for the closest species, T. mallaccensis. The scattered presence of the bacterial-like genes among distantly related eukaryotes suggests three alternative interpretations of acquisition of the novel glyoxysomal citrate synthase gene via lateral gene transfer (LGT). (1) Some eukaryotes independently acquired the gene from a common bacterium or closely related bacteria via LGT. (2) A hypothetical eukaryote once acquired the gene, which was thereafter independently transferred from the eukaryote to other eukaryotes. (3) A single event of LGT (or duplication) occurred in a certain common ancestor of eukaryotes, followed by multiple losses in many eukaryotic lineages during the subsequent evolution. Considering the monophyly of the bacterial-like eukaryotic citrate synthase genes, the first model is somewhat unlikely, even though it is not impossible. The second and third models can rationally explain the present observation, so these models are discused in some detail.     

KIN‐BASED RECOGNITION AND SOCIAL AGGREGATION IN A CILIATE

Aggregative groups entail costs that must be overcome for the evolution of complex social interactions. Understanding the mechanisms that allow aggregations to form and restrict costs of cheating can provide a resolution to the instability of social evolution. Aggregation in Tetrahymena thermophila is associated with costs of reduced growth and benefits of improved survival through “growth factor” exchange. We investigated what mechanisms contribute to stable cooperative aggregation in the face of potential exploitation by less‐cooperative lines using experimental microcosms. We found that kin recognition modulates aggregative behavior to exclude cheaters from social interactions. Long‐distance kin recognition across patches modulates social structure by allowing recruitment of kin in aggregative lines and repulsion in asocial lines. Although previous studies have shown a clear benefit to social aggregation at low population densities, we found that social aggregation has very different effects at higher densities. Lower growth rates are a cost of aggregation, but also present potential benefits when restricted to kin aggregations: slow growth and crowd tolerance allow aggregations to form and permit longer persistence on ephemeral resources. Thus in highly dynamic metapopulations, kin recognition plays an important role in the formation and stability of social groups that increase persistence through cooperative consumptive restraint.