Behavioural and morphological changes in ciliates induced by the predator Amoeba proteus

The predator Amoeba proteus induced behavioural and morphological changes in ciliates of the genus Euplotes. The frequency of avoidance behaviour in E. octocarinatus increased from 16±5% to 84±5% (SD) after 14 h of coexistence with the predator. The ciliate's width increased from 59±3 μm to 77±4 μm (SDM) within 48 h. Similar behavioural, but not morphological, change was induced in E. daidaleos, but neither morphological nor behavioural responses occurred in E. aediculatus. E. octocarinatus and E. daidaleos populations survived in the presence of A. proteus, whereas E. aediculatus populations became extinct by predation. Induced behavioural response seemed to be the reason for the low predation risk of E. octocarinatus and E. daidaleos. The results suggest that Euplotes ciliates have evolved specific defence mechanisms to various predators. Defensive changes are induced by a chemical substance released from A. proteus. This “kairomone” has a molecular weight between 5000 and 10000 Da. Proteolytic digestion of its activity indicated that the avoidance-inducing substance is a peptide. After the turbellarian Stenostomum sphagnetorum had induced a defensive morphology in E. octocarinatus or E. aediculatus, neither of these ciliates immediately avoided Amoeba proteus. Thus, Euplotes ciliates with a defensive morphology do not have behavioural defences in reaction to all predators.     

Non-lethal presence of predators modifies morphology and movement rates in <Emphasis Type="Italic">Euplotes</Emphasis>

Many species are able to modify aspects of their behaviour and morphology in the presence of predators. The aim of this study was to investigate the relationship between the expression of morphological and behavioural defences according to the framework proposed by DeWitt et al (1999). Experiments were carried out using hypotrich ciliates of the genus Euplotes as prey and turbellarians of the genus Stenostomum as predators. The smaller species Euplotes octocarinatus showed a greater proportional increase in width, a reduction in foraging movement rates and an increase in maximum movement rates following exposure to predator cues. The larger Euplotes aediculatus induced lesser changes in width, similar reductions in movement during foraging and no change in maximum speed following predator exposure. These results provide evidence of a cospecialised relationship between morphological and behavioural defences. Despite substantial differences in the absence of predators, movement rates and lateral body width were similar in both species following predator exposure. The observed changes may be considered adaptive, gape limited flatworm predators are unable to ingest large Euplotes and a reduction in movement rates during foraging reduces predator encounter rates, while an increase in maximal movement rates increases chances of predator evasion. Handling editor: S. I. Dodson     

Predation risk of typical ovoid and ‘winged’ morphs of Euplotes (Protozoa,Ciliophora)

Freshwater species of the genus Euplotes (Protozoa, Ciliophora) change their morphology in the presence of some of their predators. The ciliates develop extended lateral wings as well as dorsal and ventral projections which make engulfment by predators more difficult. In a series of laboratory experiments ingestion rates of four protozoan predators, the ciliates Lembadion bullinum, Dileptus anser, Stylonychia mytilus and Urostyla grandis, and one metazoan predator, the turbellarian Stenostomum sphagnetorum, on three species of Euplotes (E. octocarinatus, E. patella and E. aediculatus) were determined. It was calculated that the probability of rejection by a predator changed from 1:1 for ovoid morphs of Euplotes to about 2:1–20:1 for winged morphs of Euplotes, dependent on the prey and predator species that were combined. The nutritional condition of the prey also had some influence. In mixed-species cultures of prey and predators, transformed cells of E. octocarinatus survived for several months.     

The behaviour of an omnivorous protozoan affects the extent of induced morphological defence in a protozoan prey

1. The ciliate Euplotes octocarinatus responds to the presence of Stylonychia mytilus with a morphological transformation rendering its ingestion more difficult. Predation on Euplotes is reduced when an alternative prey, the flagellate Chlorogonium elongatum, is available. Lower predation is accompanied by reduced induction in Euplotes. 2. We quantified the motility of both ciliates in the presence and absence of Chlorogonium to test the hypothesis that the differential response in Euplotes is a consequence of behavioural changes affecting the encounter rate of the prey and the predator. 3. The results indicate that S. mytilus uses different feeding strategies for each prey. It is a filter feeder when small food particles (Chlorogonium) are abundant, however, it becomes a raptorial feeder when large prey with an escape capability (Euplotes) is available and small alternative prey are absent. 4. As the mobility of filter feeding Stylonychia is strongly limited, the presence of the flagellate may indirectly affect the defence level in Euplotes by reducing the frequency of contacts with the predator. 5. An experiment with Chaetogaster diastrophus, a more specialised predator not affected by the presence of Chlorogonium, as well as direct manipulation of the encounter rate by changing the surface area available for the crawling ciliates, supported the encounter rate hypothesis.     

Effects of food availability on predator-induced morphological defence in the ciliate Euplotes octocarinatus (Protista)

The effect of a prey's food resources on predator-induced morphological transformation was studied in two ciliate species: Euplotes octocarinatus (prey) and Stylonychia mytilus (predator). The ability to produce defence in Euplotes was much reduced by prolonged starvation. The extent of prey morphological transformation and therefore the effectiveness of the defence was inversely related to the length of time without food. The results suggest that the defence involves an energetic cost which may be a significant part of the whole cell budget, at least when food is in short supply. Not only the morphological transformation but also the maintenance of the anti-predator phenotype were energetically costly for Euplotes, the cost being proportional to the defence level. The morphological transformation was affected by the kind of food (algae) provided to the prey. Ciliates fed Chlorogonium elongatum attained their maximum width sooner than those fed Chlamydomonas. Thus, both the quantity and the quality of the food available may significantly affect the magnitude of response and therefore the potential success of an induced defence.     

Adaptation of inducible defense in Euplotes daidaleos (Ciliophora) to predation risks by various predators

The extent of induced morphological defense in Euplotes daidaleos correlates to this ciliate's predation risk from the defense-inducing predator species. Euplotes daidaleos responded by morphological transformation only to organisms that are able to feed on typically formed Euplotes cells (63 ± 5 m cell width in E. daidaleos). Three of those potential predator species caused defensive changes to various degrees (Student's t-test, P < 0.1 to P < 0.0001): Lembadion bullinum (Ciliata) induced 82 ± 6 m cell width in E. daidaleos; Chaetogaster diastrophus (Oligochaeta) induced 85 = 6 m width; and Stenostomum sphagnetorum (Turbellaria) induced 89 ± 8 m width (at a density of 10 predators per milliliter, respectively). At higher predator densities (50 or 100 organisms per milliliter), Euplotes developed a correspondingly larger width (to a maximum of 103 ± 10 m in the presence of S. sphagnetorum). Euplotes did not respond to organisms (e.g., Blepharisma japonicum, Colpidium campylum, Didinium nasutum, Paramecium caudatum, Spirostomum ambiguum, Stentor coeruleus) that cannot feed on this ciliate species. Daphnia longispina and Bursaria truncatella predators, which can feed on large prey of 125, or 200 m in diameter, respectively, also had no effect on the morphology of Euplotes. The extent of defense in Euplotes that was induced by 10 predators per milliliter during 24 h decreased the predation risk from those predators to 67% in the presence of S. sphagnetorum, to 50% with L. bullinum, and to 15% with C. diastrophus, compared to the typical form of Euplotes. In a natural population, the defensive form of E. daidaleos was found with average cell widths of 88 ± 8 m. The results indicate that predator-induced defense in natural Euplotes populations is beneficial to this prey and that it is adapted to the predation abilities of Euplotes predators, whereby energetical costs related to defensive changes may be saved.     

Signal-induced Defensive Phenotypic Changes in Ciliated PProtists: Morphological and Ecological Implications for Predator and Prey1

ABSTRACT. Survival of a potential prey organism depends on the effectiveness of its physical, chemical, behavioral and life history responses to the appearance of a predator. Inducible defenses are flexible responses in which predator (or competitor)-released substances stimulate potential prey organisms to transform into predator-resistant phenotypes. Induced defenses may be highly protective. Benefits however are often balanced by fitness costs such as decreased growth rates or reduced reproductive potential. Here I discuss inducible defenses in ciliates with particular attention to the hypotrich genera: Aspidisca, Euplotes, Onychodromus, Sterkiella, and an undescribed hypotrich genus. I isolated Sterkiella sp. and the undescribed genus from vernal woodland pools on Saint Anselm College campus. Experimental evidence shows that a signal-induced defensive transformation occurs in these ciliates within hours after exposure to a predator cue and results in a significant decrease in susceptibility to predation. Deployment of ciliate antipredator structures such as spines, keels, ridges and other protuberances requires a large investment of cytoskeletal elements, primarily microtubules, and incurs an evolutionary cost in the form of significantly reduced growth rates. Onychodromus quadricornutus exhibits an extraordinary degree of phenotypic plasticity. In response to different environmental conditions individuals within a clone may express one of three general phenotypes: basic, lanceolate, or giant cells. The predacious giant phenotype releases a morphogenetically active signal substance, Onychodromus-factor, that triggers defensive phenotypic transformation in both intraspecific and interspecific prey. Enzyme degradation and ultrafiltration experiments indicate that Onychodromus-factor is a peptide with a molecular weight below 10,000 Da. Conspecifics develop hypertrophied dorsal spines when exposed to Onychodromus-factor. Sterkiella cells develop two defensive dorsal keels and transform to an enlarged ovoid cell in response to Onychodromus-factor as well as inducing signals released by Stylonychia, Urosyla, and Lembadion. Field studies of two vernal pools show that defensive phenotypic transformation in Sterkiella cells coincides with the appearance of Lembadion magnum during vernal pool succession. An undescribed hypotrich genus also expresses its defended phenotype when Lembadion is present in these pools. Aspidisca turrita (Ehrenberg, 1838) Claparede and Lachmann 1858, closely resembles Aspidisca lynceus (Müller, 1773) except for the possession of a dorsal thorn-like structure. Experimental evidence shows that the dorsal thron is a defensive structure induced by signals released by the predacious ciliates Urostyla grandis and Lembadion magnum. Thus, A. turrita and A. lynceus are alternate phenotypes of the same species. I speculate that inducing signals function in predacious ciliates as lectin-like, carbohydrate-binding adhesion proteins during prey recognition and that prey species have evolved specialized cell surface receptors that allow detection of different predator proteins. I consider consequences for both predator and prey.     

A study of feeding in predacious ciliates using prey ciliates labeled with fluorescent microspheres

Feeding in predacious estuarine ciliates was investigated ina series of laboratory experiments using a new method of preylabeling which facilitates microscopic indentification of ingestedprey items. Ingestion rates of Mesodinium pulex, Euplotes vannusand E.woodruffi were estimated using the appearance, insidethe predator, of bacteriovorous ciliates (Metanophrys sp., Cyclidiumsp.and Pleuronema sp ) labeled with fluorescent microspheres. Preyremain motile and have presumably unaltered surface characteristics.Ingestion rates of log-growth phase predators increased withprey density. Mesodinium pulex ingested 0 15–0.32 cellsh^–1 over a prey concentration of 60–2300 ml^–1.Maximum ingestion rates of E. woodruffi and E. vannus were 4.5and 3.4 cells h^–1 respectively, estimated at prey abundancesof 75 and 172 cells ml^–1 respectively. Comparisons offeeding rates on prey of different sizes, and the effects ofstarvation, indicated that ingestion is likely limited by differentfactors in ‘raptorial’ (M pulex) and ‘filterfeeding’ (Euplotes spp.) predators.     

Extracellular nucleotides are active in evoking the “winged” morph of Euplotes octocarinatus

Extracellular nucleosidephosphates and -deoxyphosphates at micromolar concentrations induce Euplotes octocarinatus to change its ovoid cellshape into a “winged” morph. ATP/dATP and adenosine-tetraphosphate are most active, foUowed by CTP/dCTP, UTP/dTTP, and GTP/dGTP; ADP has less, while AMP, adenosine, cAMP, pyrophosphate and tripolyphosphate have no morphogenetic activity. Euplotes changes morphogenetically a few hours after being exposed to these compounds; changes are completed within 24–36 hr. Cell division is regularly stopped during this time. Transformed Euplotes are protected from certain predators which is reminiscent of the response triggered by predator-released proteins.     

Precise time interactions between behavioural and morphological defences

Defences induced against predators fall into three basic categories – behavioural, morphological and life history. Many species induce changes in more than one category. A theoretical advantage of a behavioural change is its potential for rapid induction compared to morphology or life history. We tested this theory by comparing modifications in behaviour and morphology along a time line in the hypotrich ciliate Euplotes octocarinatus exposed to chemical cues from Stenostomum virginianum, a predatory flatworm. Behavioural defences were induced much more rapidly than morphological, although as morphological defences became expressed changes in behaviour were slightly relaxed. This suggests a temporal compensatory relationship between the two traits. Behavioural defences are quickly induced to rapidly reduce predation risk, however they are relaxed as morphology changes are realised to avoid paying the cost of expressing both types of defence.     

Isolation of the Lembadion-factor,A morphogenetically active signal,that induces Euplotes cells to change from their ovoid form into a larger lateral winged morph

A morphogenetically active substance released by the predatory ciliate Lem-badion bullinum is recognized by ciliates of the genus Euplotes, which are potential prey organisms of Lembadion. The substance (L-factor) induces cells of the genus Euplotes to become less compact, which reduces their likelihood of becoming engulfed. Under the influence of this Lembadion- derived signal, E. octocarinatus develops extended wings and dorsal and ventral ridges and transforms within a few hours from its typical ovoid morph into an enlarged circular morph. This takes place without cell division. We have isolated the L-factor and report that it is a protein with a mass of 31,500 Da. The factor has been purified to chromatographic and electrophoretic homogeneity and was found to be active at concentrations as low as 10^?12 mol/L. © 1992 Wiley-Liss, Inc.     

How does <Emphasis Type="Italic">Euplotes</Emphasis> translation termination factor eRF1 fail to recognize the UGA stop codon?

Class 1 eukaryotic release factor 1 (eRF1) recognizes all three stop codons (UAA, UAG, and UGA) in standard-code organisms. In some ciliates with variant genetic codes, one or two stop codons are used to encode amino acids and are not recognized by eRF1; e.g., UAA and UAG are reassigned to Gln in Stylonychia and UGA is reassigned to Cys in Euplotes. Stop codon recognition is due to the N-terminal domain of eRF1 in standard-code organisms. Since variant-code ciliates most likely originate from universal-code ancestors, the N-domain sequence of their eRF1 was assumed to harbor the residues that are responsible for the changes in stop codon recognition specificity. To identify the N-domain regions determining the UGA-only specificity of Euplotes aediculatus eRF1, chimeric proteins were constructed by swapping various N-domain fragments of the E. aediculatus for their human counterparts; the MC domain was from human eRF1. Functional analysis of the chimeric eRF1 in vivo revealed two regions (residues 38–50 and 123–145) restricting the E. aediculatus eRF1 specificity to UAR. The change in stop codon recognition specificity of eRF1 was regarded as the first step in the origin of the variant genetic code in ciliates.     

The Feeding of Amoeba proteus on Paramecium aurelia*

SYNOPSIS. Density of prey (Paramecium aurelia) and predator (Amoeba proteus) were varied while volume of inorganic medium was kept constant. Variations in density of prey had little effect on the rates of feeding and reproduction of the amoebae; but with increasing predator density the amoebae captured the paramecia less rapidly and ingested fewer before dividing, altho division size did not change appreciably. Therefore, amoebae of a low density population with a constant food supply carry more nutritional reserves from generation to generation than do those in a denser population.     

Reciprocal phenotypic plasticity in a predator–prey system: inducible offences against inducible defences?

We describe one of the first examples of reciprocal phenotypic plasticity in a predator–prey system: the interaction between an inducible defence and an inducible offence. When confronted with the predatory ciliate Lembadion bullinum, the hypotrichous ciliate Euplotes octocarinatus develops protective lateral wings, which inhibit ingestion by the predator. We show that L. bullinum reacts to this inducible defence by expressing an inducible offence – a plastic increase in cell size and gape size. This counteraction reduced the effect of the defence, but did not completely neutralize it. Therefore, the defence remained beneficial for E. octocarinatus. From L. bullinum's point of view, the increase in feeding rate because of the offence was not larger than the increase in mean cell volume and apparently, did not increase the predator's fitness. Therefore, the inducible offence of L. bullinum does not seem to be an effective counter‐adaptation to the inducible defence of E. octocarinatus.     

A single amino acid change of translation termination factor eRF1 switches between bipotent and omnipotent stop-codon specificity

In eukaryotes a single class-1 translation termination factor eRF1 decodes the three stop codons: UAA, UAG and UGA. Some ciliates, like Euplotes, have a variant code, and here eRF1s exhibit UAR-only specificity, whereas UGA is reassigned as a sense codon. Since eukaryote eRF1 stop-codon recognition is associated with its N-terminal domain, structural features should exist in the N domain of ciliate eRF1s that restrict their stop-codon specificity. Using an in vitro reconstituted eukaryotic translation system we demonstrate here that a chimeric eRF1 composed of the N domain of Euplotes aediculatus eRF1 fused to the MC domains of human eRF1 exhibits UAR-only specificity. Functional analysis of eRF1 chimeras constructed by swapping Euplotes N domain sequences with the cognate regions from human eRF1 as well as site-directed mutagenesis of human eRF1 highlighted the crucial role of the alanine residue in position 70 of E. aediculatus eRF1 in restricting UGA decoding. Switching the UAR-only specificity of E. aediculatus eRF1 to omnipotent mode is due to a single point mutation. Furthermore, we examined the influence of eRF3 on the ability of chimeric and mutant eRF1s to induce peptide release in response to different stop codons.     

Immobilization of Ciliates by Concanavalin A*

SYNOPSIS. Interaction of several plant lectins with the ciliates Stylonychia mytilus, Euplotes aediculatus, and Tetrahymena pyriformis GL, was investigated. The motility of Stylonychia is specifically inhibited by treatment with concanavalin A, with which the 2 other ciliates react only weakly. Stylonychia can regain its motility by shedding the lectin-loaded surface components and rebuilding a new pellicle. Other lectins used in this study did not interact with these ciliates.     

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 15 min 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.     

Ciliate grazing on <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis> and <Emphasis Type="Italic">Nitrospira</Emphasis><Emphasis Type="Italic">moscoviensis</Emphasis>: Is selectivity a factor for the nitrogen cycle in natural aquatic systems?

Ciliated protists are important predators of bacteria in many aquatic habitats, including sediments. Since, many biochemical transformations within the nitrogen cycle are performed by bacteria, ciliates could have an indirect impact on the nitrogen cycle through selective grazing on nitrogen-transforming bacteria. As a case study, we examined ciliate grazing on nitrifying bacteria of the genera Nitrosomonas and Nitrospira. All experiments were designed as in vitro-experiments with cultures of different bacteria and ciliate species. The nitrifying bacteria used in our experiments were Nitrosomonas europaea [Winogradsky 1892] and Nitrospira moscoviensis [Ehrich 2001]. The ciliates comprised of four species that are known as efficient bacterivores and common members of the protist community in aquatic systems: Paramecium aurelia [Müller 1773], Euplotes octocarinatus [Carter 1972], Tetrahymena pyriformis [Ehrenberg 1830] and Cyclidium glaucoma [Müller 1786]. Our experimental approach, using a combination of DAPI and FISH staining, was successful in allowing the observation of ingestion of specific bacteria and their detection within ciliate food vacuoles. However, the ciliates in this study showed no significant selective grazing. No food preferences for a any bacterial taxon or any size class or morphotype were detected. Correlation with time between ciliate abundance and bacterial abundance or biovolume, using log transformed growth rates of ciliates and bacteria, showed no significant results. On the bacterial side, neither an active defence mechanism of the nitrifying bacteria against ciliate grazing, such as changes in morphology, nor competition for resources were observed. These results suggest that in our in vitro-experiments grazing by ciliates has no influence on abundance and growth of nitrifying bacteria and nitrification.     

Connection between stop codon reassignment and frequent use of shifty stop frameshifting

Ciliated protozoa of the genus Euplotes have undergone genetic code reassignment, redefining the termination codon UGA to encode cysteine. In addition, Euplotes spp. genes very frequently employ shifty stop frameshifting. Both of these phenomena involve noncanonical events at a termination codon, suggesting they might have a common cause. We recently demonstrated that Euplotes octocarinatus peptide release factor eRF1 ignores UGA termination codons while continuing to recognize UAA and UAG. Here we show that both the Tetrahymena thermophila and E. octocarinatus eRF1 factors allow efficient frameshifting at all three termination codons, suggesting that UGA redefinition also impaired UAA/UAG recognition. Mutations of the Euplotes factor restoring a phylogenetically conserved motif in eRF1 (TASNIKS) reduced programmed frameshifting at all three termination codons. Mutation of another conserved residue, Cys124, strongly reduces frameshifting at UGA while actually increasing frameshifting at UAA/UAG. We will discuss these results in light of recent biochemical characterization of these mutations.