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The escalating presence of microplastics and heavy metals in marine environments significantly jeopardizes ecological stability and human health. Despite this, research on the combined effects of microplastics/nanoplastics (MPs/NPs) and heavy metals on marine organisms remains limited. This study evaluated the impact of two sizes of polystyrene beads (approximately 2 μm and 200 nm) combined with cadmium (Cd) on the ciliate species Euplotes vannus. Results demonstrated that co-exposure of MPs/NPs and Cd markedly elevated reactive oxygen species (ROS) levels in ciliates while impairing antioxidant enzyme activities, thus enhancing oxidative damage and significantly reducing carbon biomass in ciliates. Transcriptomic profiling indicated that co-exposure of MPs/NPs and Cd potentially caused severe DNA damage and protein oxidation, as evidenced by numerous differentially expressed genes (DEGs) associated with mismatch repair, DNA replication, and proteasome function. Integrated transcriptomic and metabolomic analysis revealed that DEGs and differentially accumulated metabolites (DAMs) were significantly enriched in the TCA cycle, glycolysis, tryptophan metabolism, and glutathione metabolism. This suggests that co-exposure of MPs/NPs and Cd may reduce ciliate abundance and carbon biomass by inhibiting energy metabolism and antioxidant pathways. Additionally, compared to MPs, the co-exposure of NPs and Cd exhibited more severe negative effects due to the larger specific surface area of NPs, which can carry more Cd. These findings provide novel insights into the toxic effects of MPs/NPs and heavy metals on protozoan ciliates, offering foundational data for assessing the ecological risks of heavy metals exacerbated by MPs/NPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The application of identical exposure dosages in different species generally leads to a limited understanding of dose-response patterns because of species-specific factors. To evaluate phenol-induced ecotoxicity, antioxidant enzyme activity and population growth dynamics were compared in two model ciliates, the marine species Euplotes vannus and the freshwater species Paramecium multimicronucleatum. Dosage ranges of phenol exposure were based on tolerance limits of test ciliates as determined by their carrying capacity (K) and growth rate (r). When the exposure duration of phenol increased from 48 h to 96 h, the median effective dose (ED 50 ) for P. multimicronucleatum decreased faster than that for E. vannus , and the ratio of the former to the latter declined from 2.75 to 0.30. When E. vannus was exposed to increasing concentrations of phenol (0-140 mg l -1 ), r rose initially and then dropped significantly at concentrations higher than 40 mg l -1 , whereas K decreased linearly over the entire range. For P. multimicronucleatum, both r and K declined gradually over the range 0-200 mg l -1 phenol. Dose-response patterns of activities of three individual antioxidant enzymes, and the integrative index of the three enzymes, presented a biphasic (inverse U-shaped) curve at each of four durations of exposure, i.e. 12 h, 24 h, 36 h and 48 h. Cluster analyses and multidimensional scaling analyses of antioxidant enzyme activities revealed differences in the temporal succession of physiological states between the two model ciliates. In brief, combining ED 50 with growth dynamic parameters is helpful for designing exposure dosages of toxicants in ecotoxicity tests., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The superoxide dismutase (SOD) family is a first line antioxidant enzyme group involved in transformation of the superoxide anion (O 2 - ) into hydrogen peroxide (H 2 O 2 ) and O 2 . SOD gene expression patterns and enzyme activities therefore have a role as molecular biomarkers in evaluating the oxidative stress status of aquatic organisms. However, antioxidant enzyme systems are yet to be fully explored in the marine ciliates. In this study, we identified and characterized two types of Cu/Zn SODs (Ec-Cu/ZnSOD1 and Ec-Cu/ZnSOD2) and Ec-Mn SOD in the marine ciliate Euplotes crassus. Subsequently, SOD activity and transcriptional modulation of the relevant genes were investigated after the exposure to Cd and Cu for 8 h. All Ec-SODs showed conserved domains and metal binding sites on their active sites. Total SOD activity was induced at 1 h after exposure to Cd (125 and 1000 μg/L), and showed a marginal increase at 1-h exposure to Cu (10 and 100 μg/L). However, SOD activity was maintained at a steady level under Cd and decreased under Cu exposure conditions at 3 h and 8 h. mRNA expression of both the Ec-Cu/Zn-SODs and Mn-SOD were remarkably elevated after the exposure to Cd (250-1000 μg/L, maximum 4-fold, p < 0.05) and, in particular, Cu (25-100 μg/L, maximum > 20-fold, p < 0.05), in a concentration - dependent manner. These findings suggest that Ec-SODs may be actively involved in cellular protection against metal - mediated oxidative stress. This study is therefore helpful in understanding the molecular responses for metal toxicity in the ciliates., (Copyright © 2018 Elsevier B.V. All rights reserved.)

ATP-binding cassette (ABC) transporters participate in transporting various substances, including xenobiotics, in or out of cells. However, their genetic information and function in ciliates remain still unclear. In this study, we sequenced and characterized two ABC transporter genes (EcABCB and EcABCC), and investigated the effect of cadmium (Cd) and benzo[a]pyrene (B[a]P) on their function and gene expression, using efflux assay and real-time reverse transcription-polymerase chain reaction (qRT-PCR), respectively, in the marine ciliate, Euplotes crassus. Sequencing analysis and efflux assay showed that EcABCB and EcABCC are typical ABC transporters, possessing conserved function. Exposure to Cd (≥5mg/L) and B[a]P (≥50.5μg/L) enhanced accumulation of a substrate. A significant increase in the expression of EcABCB and EcABC mRNA was observed at lower concentration in response to Cd and B[a]P. Our findings indicate that Cd and B[a]P could inhibit the efflux function of ABC transporters, leading to cellular toxicity in the ciliate., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Understanding dose-responses is crucial for determining the utility of biomarkers in ecotoxicity assessment. Nitrofurazone is a broad-spectrum antibiotic that is widely used in the aquaculture industry in China despite its detrimental effects on ecosystems. Potential dose-response models were examined for the effect of nitrofurazone on two antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx), in the ciliated protozoan Euplotes vannus. This was achieved by measuring enzyme activity and gene expression profiling of SOD and GPx in ciliate cells exposed to nitrofurazone at doses ranging from 0 to 180mgl -1 for 6h, 12h, 18h and 24h. Dose-response dynamics were characterized by mathematical models. Results showed that: 1) dose-response patterns differed significantly among the tested endpoints, nitrofurazone concentrations and durations of exposure; 2) GPx activity was the best candidate biomarker because of its linear dose-response relationship; 3) SOD activity and mRNA relative expression levels of GPx and SOD are also candidate biomarkers but their dose-responses were non-linear and therefore more difficult to interpret; 4) partitioning the dose-response dynamic model by piecewise function can help to clarify the relationships between biological endpoints. This study demonstrates the utility of dynamic model analysis and the potential of antioxidant enzymes, in particular GPx activity, as a candidate biomarkers for environmental monitoring and risk assessment of nitrofurazone in the aquaculture industry., (Copyright © 2017. Published by Elsevier Inc.)

Marine ciliate Euplotes crassus, a single-cell eukaryote, and has been considered as a model organism for monitoring of environmental pollutions in sediments. Cytochrome P450 (CYP450) monooxygenase are phase I enzyme involved in detoxification of environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs). However, little information on CYP450 family genes in ciliate is available. In the present study, acute toxicity of PAH, benzo[a]pyrene (B[a]P) and PAH-like model compound, beta-naphthoflavone (β-NF), was investigated; full-length cDNA sequences and genomic structure of five CYP450 genes (CYP5680A1, CYP5681A1, CYP5681B1, CYP5682A1, and CYP5683A1) were analyzed; and finally their activities and transcriptional changes were measured after exposure to PAHs for 48h. According to the results, B[a]P exposure showed a negative effect on E. crassus survival, whereas β-NF exposure showed no significant effect. The 8h-LC 50 value of B[a]P was determined to be 2.449μM (95%-C.L., 7.726-3.619μM). Five genes belonging to the CYP450 family had conserved domains and clustered with those of ciliate group, as revealed in phylogenetic analysis. CYP activity did not change after exposure to B[a]P, whereas it was slightly, but significantly, induced after exposure to β-NF. The mRNA expression of five CYP450 genes was significantly modulated in a concentration- and time-dependent manner after exposure to both the chemicals. Our findings suggest that CYP450 genes in E. crassus may be involved in detoxification of B[a]P and β-NF. This study would give a better understanding about the mode of action of B[a]P and β-NF in marine ciliates at the molecular level., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The equivocality of dose-response relationships has, in practice, hampered the application of biomarkers as a means to evaluate environmental risk, yet this important issue has not yet been fully recognized or explored. This paper evaluates the potential of antioxidant enzymes in the ciliated protozoan Euplotes vannus for use as biomarkers. Dose-response dynamics, together with both the enzyme activity and the gene expression of the antioxidant enzymes, superoxide dismutase, and glutathione peroxidase, were investigated when E. vannus were exposed to graded doses of nitrofurazone for several discrete durations. Mathematical models were explored to characterize the dose-response profiles and, specifically, to identify any equivocality in terms of endpoint. Significant differences were found in both enzyme activity and messenger RNA (mRNA) expression in the E. vannus treated with nitrofurazone, and the interactions between exposure dosage and duration were significant. Correlations between enzyme activity, mRNA expression, and nitrofurazone dose varied with exposure duration. Particularly, the dose-responses showed different dynamics depending on either endpoint or exposure duration. Our findings suggest that both the enzyme activity and the gene expression of the tested antioxidant enzymes can be used as biomarkers for ecotoxicological assessment on the premise of ascertaining appropriate dosage scope, exposure duration, endpoint, etc., which can be achieved by using dose-response dynamics.

The aim of this study was to evaluate the Zn sensitivity of Euplotes vannus, Euplotes crassus, and their naturally associated bacteria sampled from sediments in the northwest and east regions of Guanabara Bay. The unexposed ciliates and bacteria did not appear to be negatively affected by 96 h of assay. In the control group, E. vannus exhibited an increase in the biomass content from 2.3 × 10(2) to 2.3 × 10(3) μg C cm(-3) between 0 and 96 h, and E. crassus increased up to 7.07 × 10(2) μg C cm(-3) at 48 h. The maximum biomass was pointed by E. crassus (1.33 × 10(3) μg C cm(-3)) in the presence of 0.005 mg Zn L(-1) and E. vannus was naturally associated bacteria (2.40 × 10(-1) μg C cm(-3)) in the presence of 1.0 mg Zn L(-1) (96 h). The growth of E. vannus from the northwest region showed concentration-dependent manners, and it is more sensitive to zinc than E. crassus from the southeast. Naturally associated bacteria showed better adaptation to increasing concentrations of Zn, and the Dunnett test showed that previous environmental selection is important. These results show that new bioremediation tools are necessary.

Trace metals and broad-spectrum antibiotic drugs are common environmental contaminants, the importance of which is increasing due to global climate change-related effects. In the present study, the biological model organism E. crassus was first acclimated to five temperatures, from 25 °C to 33 °C, followed by exposure to nominal concentrations of copper, the antibiotic model compound oxytetracycline and mixtures of both, at increasing thermal conditions. Variations of temperature-related toxicity were assessed by two high-level endpoint tests, survival and replication rates, and two sublethal parameters: endocytosis rate and lysosomal membrane stability. The selected toxicants presented opposite behaviours as the protozoa's survival rates increased following an increasing thermal gradient in the oxytetracycline-related treatments, and a decline of tolerance in metal-related treatments was observed. Results of tests combining binary mixtures of tested toxicants showed a complex pattern of responses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Euplotes crassus, a single-celled eukaryote, is directly affected by environmental contaminants. Here, exponentially cultured E. crassus were exposed to cadmium, copper, lead, and zinc and then the reactive oxygen species (ROS) and total glutathione (GSH) levels were measured. Subsequently, the transcriptional modulation of glutathione peroxidase (GPx) and glutathione reductase (GR) were estimated by quantitative RT-PCR. After an 8-h exposure, significantly higher increases in the relative ROS and total GSH levels were observed in exposed group, compared to the controls. Real-time PCR data revealed that the expression levels of GPx and GR mRNA were sensitively modulated within 8h of exposure to all heavy metals. These findings suggest that these genes may be involved in cellular defense mechanisms by modulating their gene expression against heavy metal-induced oxidative stress. Thus, they may be useful as potential molecular biomarkers to assess sediment environments for contaminants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In environmental studies, some biological responses, known as biomarkers, have been used as a powerful bioassay tool for more than four decades. Disparity between enzyme activity and mRNA abundance leads to correlation equivocality, which makes the application of biomarkers for environmental risk assessment more complicated. This study investigates this disparity in the case of catalase when used as a biomarker for detecting ecotoxicity induced by antibiotics in aquatic ecosystems. In particular, dose-responses for catalase activity and mRNA expression abundance were investigated in Euplotes vannus which were exposed to graded doses of nitrofurazone for several discrete durations, and dose-response models were developed to characterize the dose-response dynamics. Significant differences were found in both catalase activity and mRNA expression abundance among the E. vannus treated with nitrofurazone. Catalase activity showed a hormetic-like effect in terms of dose-response, characterized by a biphasic relationship which was more clearly evident after a longer exposure period, while mRNA expression abundance increased linearly with the exposure duration. Additionally, the correlation between catalase activity and mRNA expression abundance reversed along with the duration of exposure to nitrofurazone. Taken together, our results demonstrate that catalase mRNA expression offers a more straightforward dose-response model than enzyme activity. Our findings suggest that both catalase enzyme activity and mRNA expression abundance can be used jointly as bioassay tools for detecting ecotoxicity induced by nitrofurazone in aquatic ecosystems., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The taxonomic composition of phytoplankton responsible for primary production on continental shelves has changed episodically through Earth history. Geological correlations suggest that major changes in phytoplankton composition correspond in time to changes in grazing and seawater chemistry. Testing hypotheses that arise from these correlations requires experimentation, and so we carried out a series of experiments in which selected phytoplankton species were grown in treatments that differed with respect to the presence or absence of grazers as well as seawater chemistry. Both protistan (Euplotes sp.) and microarthropod (Acartia tonsa) grazers changed the growth dynamics and biochemical composition of the green alga Tetraselmis suecica, the diatom Thalassiosira weissflogii, and the cyanobacterium Synechococcus sp., increasing the specific growth rate and palatability of the eukaryotic algae, while decreasing or leaving unchanged both parameters in the cyanobacteria. Synechococcus (especially) and Thalassiosira produced toxins effective against the copepod, but ciliate growth was unaffected. Acartia induced a 4-6 fold increase of Si cell quota in the diatom, but Euplotes had no similar effect. The differential growth responses of the eukaryotic algae and cyanobacteria to ciliate grazing may help to explain the apparently coeval radiation of eukaryophagic protists and rise of eukaryotes to ecological prominence as primary producers in Neoproterozoic oceans. The experimental results suggest that phytoplankton responses to the later radiation of microarthropod grazers were clade-specific, and included changes in growth dynamics, toxin synthesis, encystment, and (in diatoms) enhanced Si uptake.

Despite an increasing number of surveys describing adverse effects of contaminated sediments on marine organisms, few studies have addressed protists. In this study, the free-crawling marine ciliate Euplotes crassus was evaluated as the test organism for the screening of sediment toxicity using sediments from both coastal and estuarine sites of the Venice Lagoon (Marghera harbour [MH], Valle Millecampi [MV], Murano island [MI] and Lido inlet [LI]). Two endpoints of high ecological value, mortality (Mry) and replication rate (RpR), were assessed in combination with the two sublethal biomarkers of stress, endocytotic rate (Ecy) and lysosomal membrane stability (NRRT). The results showed a significant inhibition of RpR, Ecy and NRRT paralleled by a small and insignificantly increased Mry of the exposed specimens. Our results thus demonstrate that only a combination of mortality and sublethal biomarkers was able to characterise an exposure-related stress syndrome. The suite of biomarkers described here was also able to detect and resolve a pollution-induced stress syndrome at an early stage of pollution. The contamination level of the sediments was assessed using chemical analysis, by estimating bioavailability and by computing a toxic pressure coefficient (TPC) to account for potential additive effects of different pollutants. The observed biological responses were consistent with the contamination levels in sediments, suggesting a high potential for using Protozoa in bioassays to assess environmental risk in coastal marine systems., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The antimicrobial activity of Cu(2)O, ZnO and NiO nanoparticles supported onto natural clinoptilolite was investigated in the secondary effluent under dark conditions. After 24h of contact the Cu(2)O and ZnO nanoparticles reduced the numbers of viable bacterial cells of Escherichia coli and Staphylococcus aureus in pure culture for four to six orders of magnitude and showed consistent 100% of antibacterial activity against native E. coli after 1h of contact during 48 exposures. The antibacterial activity of NiO nanoparticles was less efficient. The Cu(2)O and NiO nanoparticles showed 100% of antiprotozoan activity against Paramecium caudatum and Euplotes affinis after 1h of contact, while ZnO nanoparticles were less efficient. The morphology and crystallinity of the nanoparticles were not affected by microorganisms. The metal oxide nanoparticles could find a novel application in the disinfection of secondary effluent and removal of pathogenic microorganisms in the tertiary stage of wastewater treatment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The random amplified polymorphic DNA (RAPD) assay was evaluated as a potential tool to detect the ecotoxicity induced by nitrofurazone in marine ciliate, Euplotes vannus. The data revealed a reduction in viability of the test ciliates with increasing nitrofurazone concentration in the range of 0-24 mgl(-1) and time of exposure from 24 to 96 h. The nitrofurazone treated ciliates were subjected to DNA damage analysis by RAPD assay. Among the 33 test RAPD primers used in this study, 11 primers with 60-70% GC content produced unique polymorphic band patterns. A total of 213 bands of 155-3317 bp in molecular size range were observed in the untreated cells. In comparison with the control ciliates, the nitrofurazone treated groups showed differences in RAPD profiles with respect to the band intensity, disappearance of bands and appearance of new bands of amplified DNA. The variation of RAPD profiles showed both the time- and concentration-dependent relationships. The data suggested significant genomic template instability, which corresponds well with the viability of the test ciliates. Thus the results demonstrated the potential of the RAPD assay for application as a powerful tool for detecting genotoxicity induced by fishy drugs in aquatic environment., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The beaches are sites where the human influence may be strong and the beach ecosystems have often shown a high sensibility to environmental alterations. These zones may be affected by a large series of anthropogenic-derived pressures, such as unbalanced inorganic nutrient input, that may cause anomalous development of primary production, altering the structure of the trophic webs. Furthermore, the utilisation of cosmetic sunscreen products is reaching unexpected levels, thus assuming a potentially important as well as unknown role in the contamination of marine environments. The present study was planned to test the response of the beach ecosystem to increases in inorganic nutrients (nitrate and phosphate) and to the input of a widely used cosmetic sunscreen product. A short-term laboratory experiment was carried out on microsystems consisting of sediments and seawater from the swash zone of a Ligurian city beach (Sturla). The processes related to organic matter (OM) recycling and some microbial food web components (bacteria and micro-autotrophic organisms) were analysed. The multivariate statistical analysis of the results showed that the increase in inorganic nutrients and sunscreen caused only a transient alteration in the OM recycling processes in the seawater. The sedimentary processes, instead, were different in the different systems, although starting from the same condition. In the sediment, surprisingly, an increase in inorganic nutrients did not lead to an increase in the primary biomass nor to significantly higher bacterial abundance, while the sunscreen caused increased OM recycling, especially devoted to protein and lipid mobilisation, supporting a growing bacterial and autotrophic community by reducing the bottom-up pressure. Additional toxicity tests performed on protozoa highlighted that, while the inorganic nutrients seemed to show no effects, sunscreen decreased the protozoan viability, thus likely favouring microautotrophic and bacterial increases by reducing the top-down pressure., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The sesquiterpenoid euplotin C is a secondary metabolite produced by the ciliated protist Euplotes crassus and provides a mechanism for damping populations of potential competitors. Indeed, E. crassus is virtually resistant to its own product while different non-producer species representing an unbiased sample of the marine, interstitial, ciliate diversity are sensitive. For instance, euplotin C exerts a marked disruption of different homeostatic mechanisms in Euplotes vannus. We demonstrate by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay that euplotin C quickly decreases viability and mitochondrial function of E. vannus with a very high efficacy and at micromolar potency. In addition, euplotin C induces apoptosis in E. vannus as 4,6-diamino-2-phenylindole and terminal transferase dUTP nick end labeling staining show the rapid condensation and fragmentation of nuclear material in cells treated with euplotin C. These effects occur without detectable permeabilisation or rupture of cell membranes and with no major changes in the overall morphology, although some traits, such as vacuolisation and disorganized microtubules, can be observed by transmission electron microscopy. In particular, E. vannus show profound changes of the mitochondrial ultrastructure. Finally, we also show that caspase activity in E. vannus is increased by euplotin C. These data elucidate the pro-apoptotic role of euplotin C and suggest a mechanism for its impact on natural selection.

Among secondary metabolites, the acetylated hemiacetal sesquiterpene euplotin C has been isolated from the marine, ciliated protist Euplotes crassus, and provides an effective mechanism for reducing populations of potential competitors through its cytotoxic properties. However, intracellular signaling mechanisms and their functional correlates mediating the ecological role of euplotin C are largely unknown. We report here that, in E. vannus (an Euplotes morphospecies that does not produce euplotin C and shares with E. crasssus the same interstitial habitat), euplotin C rapidly increases the intracellular concentration of both Ca(2+) and Na(+), suggesting a generalized effect of this metabolite on cation transport systems. In addition, euplotin C does not induce oxidative stress, but modulates the electrical properties of E. vannus through an increase of the amplitude of graded action potentials. These events parallel the disassembling of the ciliary structures, the inhibition of cell motility, the occurrence of aberrant cytoplasmic vacuoles, and the rapid inhibition of phagocytic activity. Euplotin C also increases lysosomal pH and decreases lysosomal membrane stability of E. vannus. These results suggest that euplotin C exerts a marked disruption of those homeostatic mechanisms whose efficiency represents the essential prerequisite to face the challenges of the interstitial environment.

The ciliate, Euplotes mutabilis, isolated from industrial wastewater of tanneries of Kasur, Pakistan, showed tolerance against Cd2+ (22 microg ml(-1)), Cr6+ (60 microg ml(-1)), Pb2+ (75 microg ml(-1)) and Cu2+ (22 microg ml(-1)). The heavy metals, Cr and Pb, were randomly selected for determining the capability of the ciliate to reduce the concentration of these metal ions in the medium and to evaluate its potential use as bioremediator of wastewater. The live protozoans could remove 97% of Pb2+ and 98% of Cr6+ from the medium, 96 h after inoculation of the medium containing 10 micro gml(-1) of metal ions. The acid digestion of ciliate showed 89% of Pb2+ and 93% of Cr6+ ions accumulated in the organism. When the ciliate was exposed to heavy metals at a larger scale viz., 10 l of water containing 10 micro gml(-1) of heavy metals, it removed 86% of Pb2+ and 90% of Cr6+ from the medium. The metal uptake ability of E. mutabilis, as evidenced by its survival and growth in 100ml and 10 l of water containing 10 microg ml(-1) of metal ions, reduction in the concentration of heavy metals in the medium and its increased uptake by the live cells, and no metal uptake by the heat killed ciliate can be exploited for metal detoxification of industrial wastes and environmental clean-up operations.

It is now widely accepted that assays with protists are relevant to be exploited for the study of environmental modifications due to the presence of xenobiotic compounds. In this work, the possibility of utilizing Euplotes crassus, an interstitial marine ciliate, for the pre-chemical screening of estuarine and coastal sediments was evaluated. For this purpose, the effects of exposure to pollutants were tested on the cell viability, fission rate and lysosomal membrane stability of E. crassus. The following toxicants were used: an organophosphate (OP) pesticide, basudin, an organochlorine hydrocarbon, AFD25, both employed especially for pest control in agricultural sites, a toxic heavy metal, mercury (HgCl2) and different mixtures of the above-mentioned compounds, as they might occur in polluted sites. Exposure to these toxicants affected cell viability at concentrations ranging from 96.6 to 966 x 10(3)mg/l for basudin, from 3.3 to 33 x 10(3)mg/l for AFD25 and from 0.1 to 1mg/l for HgCl2. A significant decrease in the mean fission rate (P<0.001) was found after 24- or 48-h exposures to 9.66 mg/l basudin, 3.3 mg/l AFD25 and 7 x 10(-2)mg/l HgCl2. Furthermore, the Neutral Red Retention Assay showed a significant decrease in lysosomal membrane stability after 60- and 120-min exposures to AFD25 (33 mg/l) and HgCl2 (0.33 mg/l). In addition, as it is well-known that the inhibition of acetylcholinesterase activity represents a specific biomarker of exposure to OP and carbamate pesticides in higher organisms, initially the presence of cholinesterase (ChE) activity was detected in E. crassus, using cytochemical, spectrophotometric and electrophoretic methods. Afterwards, this enzyme activity was characterized spectrophotometrically by its sensitivity to specific ChE inhibitors and to variations in pH and temperature. The ChE activity was inhibited significantly by basudin- (9.66 and 96.6 mg/l) or AFD25-exposure (3.3 mg/l). Conversely, exposure to AFD25 (33 mg/l) or HgCl2 (0.1 and 0.3mg/l) caused a significant increase in this enzyme activity. Moreover, exposure to mixtures containing basudin, AFD25 and HgCl2 was found to affect the cell viability, the mean fission rate and the ChE activity differently, in an unpredictable manner. Our results indicate that E. crassus seems to be a suitable test organism to evaluate the toxicity of marine sediments.

The hypotrichous ciliated protozoan Euplotes aediculatus possesses a characteristic C-shaped somatic nucleus (macronucleus) within the cytoplasm, which shows dynamic shape change during the cell cycle. It is shown that isolated macronuclei possess Ca(2+)-dependent contractility. Macronuclei were isolated, stuck fast on the glass surface, and subjected to different concentrations of Ca(2+) in a Ca(2+)-EGTA buffer. The nuclei became expanded at [Ca(2+)]<10(-7)M, and they contracted on subsequent addition of higher concentrations of Ca(2+). Cycles of expansion and contraction of the nucleus could be repeated many times by alternate addition of EGTA and Ca(2+), indicating that the size of isolated nuclei can be regulated by [Ca(2+)] alone. The nuclear contraction was observed in all phases of the cell cycle, but contractility was less evident around replication bands in the S phase. In addition to the hypotrichous ciliate Euplotes, similar Ca(2+)-dependent nuclear contractility was found to exist in other cell types, including protozoans of different taxa (a heliozoon Actinophrys sol and a peniculine ciliate Paramecium bursaria), and also mammalian culture cells (HeLa cells). Our findings suggest a possibility that Ca(2+)-dependent nuclear contractility may be shared among diverse eukaryotic organisms.

Telomerase is a unique ribonucleoprotein that reverse transcribes a defined region of its RNA subunit onto the ends of eukaryotic chromosomes. The product of telomerase, telomeric DNA, is typically a G-rich repeated sequence, (TTTTGGGG)(n) in the ciliate Euplotes aediculatus and (TTAGGG)(n) in humans. Telomerase can extend oligonucleotide primers in vitro in a processive fashion. We used dNTP analogues to study the structure-activity relationship between substrate nucleotides and processivity of telomerase from E. aediculatus. Several analogues, including 2'-deoxyuridine triphosphate (dUTP), 2'-deoxyinosine triphosphate (dITP), and 7-deaza-2'-deoxyguanosine triphosphate (7-deaza-dGTP), were good substrates for telomerase with K(m) and V(max) values near those of the natural substrates, dTTP and dGTP. However, telomerase processivity was affected with these substrates, decreasing in the order dUTP > 7-deaza-dGTP > dITP. Telomerase did not completely reverse transcribe the template when dITP was the substrate, and it efficiently extended a primer by the addition of two repeats when 7-deaza-dGTP and dUTP were utilized. When the same nucleotide analogues were incorporated into the primers, no effects were observed except in the case of a 3'-terminal deoxyinosine. The data support a model that includes the formation of an intramolecular secondary structure within the product DNA to facilitate translocation. The most likely structure is a G-G hairpin.

Macronuclear chromosomes of hypotrichous ciliates are gene-sized molecules carrying the coding sequence flanked by short non-translated regions and bounded by telomeres. We have constructed artificial chromosomes for investigation of transcription in the macronucleus of Euplotes crassus. The neo gene was put under the control of the 5"-non-translated region of the TBP gene of E.crassus. These molecules were introduced into the cell with the help of liposomes. The cells were transformed and survived high concentrations of geneticin. The artificial chromosomes were kept in the macro-nucleus for at least 50 days at a copy number of about 200 per macronucleus. Expression of the gene was shown by reverse transcription of the neo messenger. The transcription start was mapped and found to coincide with that found on the natural macronuclear chromosome encoding TBP in E.crassus.

We have cloned the macronuclear linear DNA molecule carrying the ribosomal RNA genes from the ciliated protozoan Euplotes crassus. DNA sequence analysis was carried out to locate coding regions and to determine whether sequences that have been mutated to confer antibiotic resistance are conserved in the E. crassus genes. The beginning and end of the primary transcript were mapped. In order to determine whether conserved sequences that might serve as replication origins were present, the 5' and 3' non-coding sequences from E. crassus were compared to the corresponding sequences from the macronuclear linear rDNA molecules from the following euplotid species: Euplotes vannus, Euplotes minuta, Euplotes raikovii and Euplotes rariseta. A DNA transformation construct was made by generating a putative anisomycin resistant mutation along with a mutation generating a restriction site polymorphism. Microinjection of the construct into the developing macronucleus of mated cells resulted in exconjugant cell lines with increased resistance to anisomycin. The injected rDNA with the restriction site polymorphism is detectable in the anisomycin resistant cells and appears to represent a minor fraction of the rDNA.

As a first step towards developing a DNA transformation method for the ciliated protozoan Euplotes crassus we determined the minimum inhibitory concentration (MIC) for cell division in the presence of cycloheximide (Chx) for several cell lines and the range of Chx sensitivity for 106 different progeny cell lines derived by mating two lines. All of the cell lines are highly sensitive to Chx. Progeny cell lines show a wider range of sensitivities than the parental lines. Because site-directed mutagenesis of the RPL29 gene encoding the large subunit ribosomal protein 29 (L29) has been used to generate a Chx-resistance marker (ChxR) for another ciliate, Tetrahymena thermophila [Yao and Yao, Proc. Natl. Acad. Sci. USA 88 (1991) 9493-9497], we isolated and sequenced the entire E. crassus macronuclear DNA carrying RPL29. The encoded peptide is 52-73% identical in sequence to L29 sequences from organisms ranging from T. thermophila and Saccharomyces cerevisiae to mouse. In E. crassus, the codon that has been mutated to confer Chx resistance in both S. cerevisiae and T. thermophila already encodes the amino-acid residue of one of the mutant forms identified in these other organisms. Thus, E. crassus RPL29 is not a convenient source of a selectable marker. Notable features of the macronuclear DNA carrying RPL29 are its extremely short non-coding regions and a TAG stop codon.