Coupling between Ribotypic and Phenotypic Traits of Protists across Life Cycle Stages and Temperatures.

Relationships between ribotypic and phenotypic traits of protists across life cycle stages remain largely unknown. Herein, we used single cells of two soil and two marine ciliate species to examine phenotypic and ribotypic traits and their relationships across lag, log, plateau, cystic stages and temperatures. We found that Colpoda inflata and Colpoda steinii demonstrated allometric relationships between 18S ribosomal DNA (rDNA) copy number per cell (CNPC), cell volume (CV), and macronuclear volume across all life cycle stages. Integrating previously reported data of Euplotes vannus and Strombidium sulcatum indicated taxon-dependent rDNA CNPC-CV functions. Ciliate and prokaryote data analysis revealed that the rRNA CNPC followed a unified power-law function only if the rRNA-deficient resting cysts were not considered. Hence, a theoretical framework was proposed to estimate the relative quantity of resting cysts in the protistan populations with total cellular rDNA and rRNA copy numbers. Using rDNA CNPC was a better predictor of growth rate at a given temperature than rRNA CNPC and CV, suggesting replication of redundant rDNA operons as a key factor that slows cell division. Single-cell high-throughput sequencing and analysis after correcting sequencing errors revealed multiple rDNA and rRNA variants per cell. Both encystment and temperature affected the number of rDNA and rRNA variants in several cases. The divergence of rDNA and rRNA sequence in a single cell ranged from 1% to 10% depending on species. These findings have important implications for inferring cell-based biological traits (e.g., species richness, abundance and biomass, activity, and community structure) of protists using molecular approaches. IMPORTANCE Based on phenotypic traits, traditional surveys usually characterize organismal richness, abundance, biomass, and growth potential to describe diversity, organization, and function of protistan populations and communities. The rRNA gene (rDNA) and its transcripts have been widely used as molecular markers in ecological studies of protists. Nevertheless, the manner in which these molecules relate to cellular (organismal) and physiological traits remains poorly understood, which could lead to misinterpretations of protistan diversity and ecology. The current research highlights the dynamic nature of cellular rDNA and rRNA contents, which tightly couple with multiple phenotypic traits in ciliated protists. We demonstrate that quantity of resting cysts and maximum growth rate of a population can be theoretically estimated using ribotypic trait-based models. The intraindividual sequence polymorphisms of rDNA and rRNA can be influenced by encystment and temperature, which should be considered when interpreting species-level diversity and community structure of microbial eukaryotes.

Evaluation of phenol-induced ecotoxicity in two model ciliate species: Population growth dynamics and antioxidant enzyme activity.

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 (ED50) 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 ED50 with growth dynamic parameters is helpful for designing exposure dosages of toxicants in ecotoxicity tests.

New contribution to the species-rich genus Euplotes: Morphology, ontogeny and systematic position of two species (Ciliophora; Euplotia).

The morphology, ontogeny and phylogeny of two Euplotes species, E. estuarinus sp. nov. and a population of E. platystoma Dragesco and Dragesco-Kernéis, 1986, both collected from tropical brackish waters in south China, were investigated based on living morphology, ciliary pattern and molecular data. Euplotes estuarinus sp. nov. is small (about 60 × 40 µm in vivo), has a dargyrome of the double-eurystomus type, and the transverse cirri are arranged in two groups, with two left and three right ones. The original description of the poorly known species, E. platystoma, is brief, and the species was never investigated using live observation and molecular methods Hence, we provided a detailed redescription. Some stages of their morphogenesis were observed which proceed in the same pattern as in their congeners. The new species E. estuarinus sp. nov. clusters with E. curdsi, differing only by 1 bp in their SSU rRNA gene sequences, which is likely due to the recent speciation event and the limited resolution of the SSU rRNA gene at species level in this group as the two species are clearly morphologically distinct.

Morphological and Molecular Redefinition of Euplotes platystoma Dragesco & Dragesco-Kernéis, 1986 and Aspidisca lynceus (Müller,1773) Ehrenberg, 1859, with Reconsideration of a "Well-known" Euplotes Ciliate, Euplotes harpa Stein, 1859 (Ciliophora, Euplotida).

We documented the morphology, infraciliature, silverline system, and molecular data of two euplotid species isolated from China, including two populations of the poorly known Euplotes platystoma Dragesco & Dragesco-Kernéis, and the previously well described Aspidisca lynceus (Müller, ) Ehrenberg, 1830. Based on the information available, an improved diagnosis of Euplotes platystoma is given, including: a narrow adoral zone with 44-68 membranelles, 10 frontoventral, 5 transverse, 2 left marginal and 2 caudal cirri, 11-13 dorsal kineties with 17-25 dikinetids in the mid-dorsal row, and dorsal silverline system of the double-eurystomus type. The Chinese population of Aspidisca lynceus closely resembles previously described populations. Phylogenetic analyses inferred from SSU rDNA sequences show that E. platystoma is closely related with E. neapolitanus, and the internal position of A. lynceus within this genus is still not robust. A reconsideration of the "well-known" Euplotes harpa and a comparison of all SSU rDNA sequences of E. harpa in GenBank are provided. We speculate that the sequences available from GenBank under the name of E. harpa are very likely from misidentified materials, that is, the identity of the species currently associated with the SSU rDNA of this "well-known" form in molecular databases requires further confirmation.

Identification and molecular characterization of cytochrome P450 (CYP450) family genes in the marine ciliate Euplotes crassus: The effect of benzo[a]pyrene and beta-naphthoflavone.

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 (ß-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 ß-NF exposure showed no significant effect. The 8h-LC50 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 ß-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 ß-NF. This study would give a better understanding about the mode of action of B[a]P and ß-NF in marine ciliates at the molecular level.

Did Gause Have a Yeast Infection?

We planned to develop predator-prey models using Paramecium and yeast, but they have not been empirically examined since work by Gause in the 1930s. Therefore, we evaluated if Paramecium aurelia ingests and grows on eight yeasts. Recognising that it ingested yeasts but could not grow, we assessed if it might grow on other yeasts, by empirically parameterising a predator-prey model that relies on ingestion, not growth. Simulations were compared to P. aurelia-yeast time-series data, from Gause. We hypothesised that if the model simulated predator-prey dynamics that mimicked the original data, then possibly P. aurelia could grow on yeast; simulations did not mimic the original data. Reviewing works by Gause exposed two issues: experiments were undoubtedly contaminated with bacteria, allowing growth on bacteria, not yeast; and the population cycle data cannot be considered a self-sustaining time series, as they were manipulated by adding yeast and ciliates. We conclude that past and future work should not rely on this system, for either empirical or theoretical evaluations. Finally, although we show that P. aurelia, P. caudatum, Euplotes patella, and Blepharisma sp. cannot grow on yeast, Tetrahymena pyriformis and Colpidium striatum can; these may provide models to explore predator-prey dynamics.

Proteomic approach to reveal the proteins associated with encystment of the ciliate Euplotes encysticus.

In order to identify and reveal the proteins related to encystment of the ciliate Euplotes encysticus, we analyzed variation in the abundance of the proteins isolated from the resting cyst comparing with proteins in the vegetative cell. 2-D electrophoresis, MALDI-TOF MS techniques and Bioinformatics were used for proteome separation, quantification and identification. The comparative proteomics studies revealed 26 proteins with changes on the expression in the resting cysts, including 12 specific proteins and 14 differential proteins. 12 specific proteins and 10 out of the 14 differential proteins were selected and identified by MALDI-TOF MS. The identified specific proteins with known functions included type II cytoskeletal 1, keratin, Nop16 domain containing protein, protein arginine n-methyltransferase, epsilon-trimethyllysine hydroxylase and calpain-like protein. The identified differential proteins with known functions included Lysozyme C, keratinocyte growth factor, lysozyme homolog AT-2, formate acetyltransferase, alpha S1 casein and cold-shock protein. We discussed the functions of these proteins as well as their contribution in the process of encystment. These identified proteins covered a wide range of molecular functions, including gene regulation, RNA regulation, proteins degradation and oxidation resistance, stress response, material transport and cytoskeleton organization. Therefore, differential expression of these proteins was essential for cell morphological and physiological changes during encystment. This suggested that the peculiar proteins and differential proteins might play important roles in the process of the vegetative cells transforming into the resting cysts. These observations may be novel findings that bring new insights into the detailed mechanisms of dormancy.

Grazers and phytoplankton growth in the oceans: an experimental and evolutionary perspective.

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.

Ciliate-adenovirus interactions in experimental co-cultures of Euplotes octocarinatus and in wastewater environment.

The aim of the present work was to study the dynamics of the interactions between human adenovirus and ciliates under both experimental and field conditions. Experimental co-cultures of the ciliated protozoan Euplotes octocarinatus and human adenovirus (HAdV) type 2 were established and virus internalization was investigated using nested PCR and direct immunofluorescence (IF). In addition, to study protozoa-virus interactions in the field, wild ciliates were isolated from active sludges of a wastewater treatment plant and analyzed for the presence of adenovirus using direct IF. In vitro experiments revealed HAdV type 2 inside Euplotes cells after 15min of contact and its persistence until at least 35 days post infection. In addition, our results showed the adsorption of adenovirus on the surface of wild ciliates. We conclude that HAdV is taken up by ciliates, however more studies are necessary in order to better investigate the mechanisms, the infectivity of internalized virus and the protective effects of internalization against disinfection.

The use of protozoa in ecotoxicology: application of multiple endpoint tests of the ciliate E. crassus for the evaluation of sediment quality in coastal marine ecosystems.

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.

Divergences in the response to ultraviolet radiation between polar and non-polar ciliated protozoa: UV radiation effects in Euplotes.

Ultraviolet (UV) radiation has detrimental effects on marine ecosystems, in particular in the polar regions where stratospheric ozone reduction causes higher levels of solar radiation. We analyzed two polar species of Euplotes, Euplotes focardii and Euplotes nobilii, for the sensitivity to UV radiation in comparison with two akin species from mid-latitude and tropical waters. Results showed that they face UV radiation much more efficiently than the non-polar species by adopting alternative strategies that most likely reflect different times of colonization of the polar waters. While E. focardii, which is endemic to the Antarctic, survives for longer exposed to UV radiation, E. nobilii, which inhabits both the Antarctic and Arctic, recovers faster from UV-induced damage.

Did sulfate availability facilitate the evolutionary expansion of chlorophyll a+c phytoplankton in the oceans?

During the Mesozoic Era, dinoflagellates, coccolithophorids and diatoms became prominent primary producers in the oceans, succeeding an earlier biota in which green algae and cyanobacteria had been proportionally more abundant. This transition occurred during an interval marked by increased sulfate concentration in seawater. To test whether increasing sulfate availability facilitated the evolutionary transition in marine phytoplankton, the cyanobacterium Synechococcus sp., the green alga Tetraselmis suecica and three algae containing chlorophyll a+c (the diatom Thalassiosira weissflogii, the dinoflagellate Protoceratium reticulatum and the coccolithophorid Emiliania huxleyi) were grown in media containing 1, 5, 10, 20, or 30 mm SO(4) (2-) . The cyanobacterium and the green alga showed no growth response to varying [SO(4) (2-) ]. By contrast, the three chlorophyll a+c algae showed improved growth with higher [SO(4) (2-) ], but only up to 10 mm. The chlorophyll a+c algae, but not the green alga or cyanobacterium, also showed lower C:S with higher [SO(4) (2-) ]. When the same experiment was repeated in the presence of a ciliate predator (Euplotes sp.), T. suecica and T. weissflogii increased their specific growth rate in most treatments, whereas the growth rate of Synechococcus sp. was not affected or decreased in the presence of grazers. In a third experiment, T. suecica, T. weissflogii, P. reticulatum and Synechococcus sp. were grown in conditions approximating modern, earlier Paleozoic and Proterozoic seawater. In these treatments, sulfate availability, nitrogen source, metal availability and Pco(2) varied. Monospecific cultures exhibited their highest growth rates in the Proterozoic treatment. In mixed culture, T. weissflogii outgrew other species in modern seawater and T.suecica outgrew the others in Paleozoic water. Synechococcus sp. grew best in Proterozoic seawater, but did not outgrow eukaryotic species in any treatment. Collectively, our results suggest that secular increase in seawater [SO(4) (2-) ] may have facilitated the evolutionary expansion of chlorophyll a+c phytoplankton, but probably not to the exclusion of other biological and environmental factors.

Isolation and structural characterization of two water-borne pheromones from Euplotes crassus, a ciliate commonly known to carry membrane-bound pheromones.

Ciliates comprise species synthesizing water-diffusible mating type factors or pheromones and species synthesizing insoluble, cell membrane-bound pheromones. Euplotes crassus has traditionally been placed in the latter group. In contrast with this notion, we found that E. crassus is a constitutive pheromone-secreting ciliate, like other Euplotes species. From cell-free filtrate preparations of the E. crassus strain L-2D, we isolated two distinct pheromones, designated as Ec-α and Ec-1, and determined their complete amino acid sequences by combined chemical and genetic approaches. The Ec-α pheromone sequence extends for 56 amino acid residues with six cysteines and shows a molecular mass of 6,183 Da, while the Ec-1 pheromone sequence extends for 45 amino acid residues with 10 cysteines and shows a molecular mass of 4,840 Da. Marked structural differences distinguish the full-length Ec-α and Ec-1 coding sequences, which have been cloned and characterized from the transcriptionally active macronuclear genome. They were taken as clear indication that the Ec-α and Ec-1 pheromones are specified by genes that are not allelic, but likely derived from a duplicated genetic locus of the transcriptionally silent micronuclear genome.

Organic matter recycling in a beach environment influenced by sunscreen products and increased inorganic nutrient supply (Sturla, Ligurian Sea, NW Mediterranean).

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.

Heavy metal uptake by Euplotes mutabilis and its possible use in bioremediation of industrial wastewater.

A ciliate protozoan, Euplotes mutabilis, isolated from heavy metal laden industrial wastewater, has been shown to tolerate multiple heavy metals thus suggesting its significance in bioremediation of industrial effluents. This ciliate tolerated Zn(2+) up to 33 microg/mL, Cd(2+) up to 22 microg/mL and Ni(2+) up to 18 microg/mL. The ciliate could uptake 85% Zn(2+), 84% of Cd(2+) and 87% of Ni(2+) after 96 h of inoculation of growth medium containing 10 microg/mL of Zn(2+) and 5 microg/mL of Cd(2+) and Ni(2+), with actively growing ciliates. After 6 days of incubation the ciliate removed 87% Cd(2+), 92% Ni(2+), and 93% Zn(2+) from the wastewater. The heavy metal uptake capability of Euplotes mutabilis may be employed for metal detoxification operations.

Euplotespora binucleata n. gen., n. sp. (Protozoa: Microsporidia), a parasite infecting the hypotrichous ciliate Euplotes woodruffi, with observations on microsporidian infections in ciliophora.

A new microsporidian species, Euplotespora binucleata n. gen., n. sp., from the brackish-water ciliate Euplotes woodruffi is described and defined on the basis of life history characteristics, light and electron microscopic features, and small subunit (SSU) ribosomal DNA (rDNA) sequencing. The life cycle of E. binucleata n. sp. probably has rather short merogonic and relatively long sporogonic phases. Some uninuclear meronts and sporonts, along with diplokaryotic sporoblasts and spores, were found in experimentally infected host cells. Such a peculiar life cycle has been induced experimentally in Euplotes eurystomus and constitutively microsporidian-free stocks of E. woodruffi. Spores of E. binucleata n. sp. are monomorphic, ovoid-cylindrical in shape, 3.44+/-0.17 x 1.65+/-0.22 microm in size, and characterized by a diplokaryotic condition and a large posterior vacuole. The polar tube is isofilar, 4.5-5.5 microm in length when ejected, and lacking a distinctive coiled region (half-coiled). The polaroplast is divided into two regions: the anterior part has a few lamellae close to the anchoring disc; and the posterior part is a rounded body (sack), about one-quarter of the spore length. Spores do not appear to cluster together as a group. Each spore is surrounded by a sporophorous membrane closely adjacent to the exospore layer. A phylogenetic analysis of SSU rDNA sequences by different methods placed E. binucleata n. sp. in a clade with representatives of the microsporidian genera Cystosporogenes and Vittaforma. Observations of microsporidia in several other ciliates are discussed in view of the microsporidian infection frequency in the phylum Ciliophora.

Heavy metal resistant freshwater ciliate, Euplotes mutabilis, isolated from industrial effluents has potential to decontaminate wastewater of toxic metals.

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.

Sequencing of random Euplotes crassus macronuclear genes supports a high frequency of +1 translational frameshifting.

Programmed translational frameshifts have been identified in genes from a broad range of organisms, but typically only a very few genes in a given organism require a frameshift for expression. In contrast, a recent analysis of gene sequences available in GenBank from ciliates in the genus Euplotes indicated that >5% required one or more +1 translational frameshifts to produce their predicted protein products. However, this sample of genes was nonrandom, biased, and derived from multiple Euplotes species. To test whether there truly is an abundance of frameshift genes in Euplotes, and to more accurately assess their frequency, we sequenced a random sample of 25 cloned genes/macronuclear DNA molecules from Euplotes crassus. Three new candidate +1 frameshift genes were identified in the sample that encode a membrane occupation and recognition nexus (MORN) repeat protein, a C(2)H(2)-type zinc finger protein, and a Ser/Thr protein kinase. Reverse transcription-PCR analyses indicate that all three genes are expressed in vegetatively proliferating cells and that the mRNAs retain the requirement of a frameshift. Although the sample of sequenced genes is relatively small, the results indicate that the frequency of genes requiring frameshifts in E. crassus is between 3.7% and 31.7% (at a 95% confidence interval). The current and past data also indicate that frameshift sites are found predominantly in genes that likely encode nonabundant proteins in the cell.

Tec3, a new developmentally eliminated DNA element in Euplotes crassus.

More than 100,000 interstitial segments of DNA (internal eliminated sequences [IESs]) are excised from the genome during the formation of a new macronucleus in Euplotes crassus. IESs include unique sequence DNA as well as two related families of transposable elements, Tec1 and Tec2. Here we describe a new class of E. crassus transposons, Tec3, which is present in 20 to 30 copies in the micronuclear genome. Tec3 elements have long inverted terminal repeats and contain a degenerate open reading frame encoding a tyrosine-type recombinase. One characterized copy of Tec3 (Tec3-1) is 4.48 kbp long, has 1.23-kbp inverted terminal repeats, and resides within the micronuclear copy of the ribosomal protein L29 gene (RPL29). The 23 bp at the extreme ends of this element are very similar to those in other E. crassus IESs and, like these other IESs, Tec3-1 is excised during the polytene chromosome stage of macronuclear development to generate a free circular form with an unusual junction structure. In contrast, a second cloned element, Tec3-2, is quite similar to Tec3-1 but lacks the terminal 258 bp of the inverted repeats, so that its ends do not resemble the other E. crassus IES termini. The Tec3-2 element appears to reside in a large segment of the micronuclear genome that is subject to developmental elimination. Models for the origins of these two types of Tec3 elements are presented, along with a discussion of how some members of this new transposon family may have come to be excised by the same machinery that removes other E. crassus IESs.