Phenotypic plasticity and the effects of thermal fluctuations on specialists and generalists.

Classical theories predict that relatively constant environments should generally favour specialists, while fluctuating environments should be selected for generalists. However, theoretical and empirical results have pointed out that generalist organisms might, on the contrary, perform poorly under fluctuations. In particular, if generalism is underlaid by phenotypic plasticity, performance of generalists should be modulated by the temporal characteristics of environmental fluctuations. Here, we used experiments in microcosms of Tetrahymena thermophila ciliates and a mathematical model to test whether the period or autocorrelation of thermal fluctuations mediate links between the level of generalism and the performance of organisms under fluctuations. In the experiment, thermal fluctuations consistently impeded performance compared with constant conditions. However, the intensity of this effect depended on the level of generalism: while the more specialist strains performed better under fast or negatively autocorrelated fluctuations, plastic generalists performed better under slow or positively autocorrelated fluctuations. Our model suggests that these effects of fluctuations on organisms' performance may result from a time delay in the expression of plasticity, restricting its benefits to slow enough fluctuations. This study points out the need to further investigate the temporal dynamics of phenotypic plasticity to better predict its fitness consequences under environmental fluctuations.

Quantitative proteomic analyses of a Pb-adapted Tetrahymena thermophila strain reveal the cellular strategy to Pb(II) stress including lead biomineralization to chloropyromorphite.

A strain of the protozoan ciliate Tetrahymena thermophila adapted to increasing Pb(II) concentrations over two years has shown that one of the resistance mechanisms to this extreme metal stress is the lead biomineralization to chloropyromorphite, one of the most stable minerals in the earth's crust. Several techniques such as microanalysis coupled to transmission and scanning electron microscopy (X-Ray Energy Disperse Spectroscopy), fluorescence microscopy and X-ray power diffraction analysis have revealed the presence of chloropyromorphite as crystalline aggregates of nano-globular structure, together with the presence of other secondary lead minerals. This is the first time that the existence of this type of biomineralization in a ciliate protozoan is described. The Pb(II) bioremediation capacity of this strain has shown that it can remove >90 % of the toxic soluble lead from the medium. A quantitative proteomic analysis of this strain has revealed the main molecular-physiological elements involved in adaptation to Pb(II) stress: increased activity of proteolytic systems against lead proteotoxicity, occurrence of metallothioneins to immobilize Pb(II) ions, antioxidant enzymes to mitigate oxidative stress, and an intense vesicular trafficking presumably involved in the formation of vacuoles where pyromorphite accumulates and is subsequently excreted, together with an enhanced energy metabolism. As a conclusion, all these results have been compiled into an integrated model that could explain the eukaryotic cellular response to extreme lead stress.

The Tetrahymena bcd1 mutant implicates endosome trafficking in ciliate, cortical pattern formation.

Ciliates, such as Tetrahymena thermophila, evolved complex mechanisms to determine both the location and dimensions of cortical organelles such as the oral apparatus (OA: involved in phagocytosis), cytoproct (Cyp: for eliminating wastes), and contractile vacuole pores (CVPs: involved in water expulsion). Mutations have been recovered in Tetrahymena that affect both the localization of such organelles along anterior-posterior and circumferential body axes and their dimensions. Here we describe BCD1, a ciliate pattern gene that encodes a conserved Beige-BEACH domain-containing protein a with possible protein kinase A (PKA)-anchoring activity. Similar proteins have been implicated in endosome trafficking and are linked to human Chediak-Higashi syndrome and autism. Mutations in the BCD1 gene broaden cortical organelle domains as they assemble during predivision development. The Bcd1 protein localizes to membrane pockets at the base of every cilium that are active in endocytosis. PKA activity has been shown to promote endocytosis in other organisms, so we blocked clathrin-mediated endocytosis (using "dynasore") and inhibited PKA (using H89). In both cases, treatment produced partial phenocopies of the bcd1 pattern mutant. This study supports a model in which the dimensions of diverse cortical organelle assembly-platforms may be determined by regulated balance between constitutive exocytic delivery and PKA-regulated endocytic retrieval of organelle materials and determinants.

Population dynamics hide phenotypic changes driven by subtle chemical exposures: implications for risk assessments.

Ecological risk assessment of chemicals focuses on the response of different taxa in isolation not taking ecological and evolutionary interplay in communities into account. Its consideration would, however, allow for an improved assessment by testing for implications within and across trophic levels and changes in the phenotypic and genotypic diversity within populations. We present a simple experimental system that can be used to evaluate the ecological and evolutionary responses to chemical exposure at microbial community levels. We exposed a microbial model system of the ciliate Tetrahymena thermophila (predator) and the bacterium Pseudomonas fluorescens (prey) to iron released from Magnetic Particles (MP-Fedis), which are Phosphorus (P) adsorbents used in lake restoration. Our results show that while the responses of predator single population size differed across concentrations of MP-Fedis and the responses of prey from communities differed also across concentration of MP-Fedis, the community responses (species ratio) were similar for the different MP-Fedis concentrations. Looking further at an evolutionary change in the bacterial preys' defence, we found that MP-Fedis drove different patterns and dynamics of defence evolution. Overall, our study shows how similar community dynamics mask changes at evolutionary levels that would be overlooked in the design of current risk assessment protocols where evolutionary approaches are not considered.

Ccdc113/Ccdc96 complex, a novel regulator of ciliary beating that connects radial spoke 3 to dynein g and the nexin link.

Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern.

Shulin packages axonemal outer dynein arms for ciliary targeting.

The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During ciliogenesis, these ~1.8-megadalton complexes are assembled in the cytoplasm and targeted to cilia by an unknown mechanism. Here, we used the ciliate Tetrahymena to identify two factors (Q22YU3 and Q22MS1) that bind ODAs in the cytoplasm and are required for ODA delivery to cilia. Q22YU3, which we named Shulin, locked the ODA motor domains into a closed conformation and inhibited motor activity. Cryo-electron microscopy revealed how Shulin stabilized this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia.

Evolution under pH stress and high population densities leads to increased density-dependent fitness in the protist Tetrahymena thermophila.

Abiotic stress is a major force of selection that organisms are constantly facing. While the evolutionary effects of various stressors have been broadly studied, it is only more recently that the relevance of interactions between evolution and underlying ecological conditions, that is, eco-evolutionary feedbacks, have been highlighted. Here, we experimentally investigated how populations adapt to pH-stress under high population densities. Using the protist species Tetrahymena thermophila, we studied how four different genotypes evolved in response to stressfully low pH conditions and high population densities. We found that genotypes underwent evolutionary changes, some shifting up and others shifting down their intrinsic rates of increase (r0 ). Overall, evolution at low pH led to the convergence of r0 and intraspecific competitive ability (α) across the four genotypes. Given the strong correlation between r0 and α, we argue that this convergence was a consequence of selection for increased density-dependent fitness at low pH under the experienced high density conditions. Increased density-dependent fitness was either attained through increase in r0 , or decrease of α, depending on the genetic background. In conclusion, we show that demography can influence the direction of evolution under abiotic stress.

Variability in Dispersal Syndromes Is a Key Driver of Metapopulation Dynamics in Experimental Microcosms.

Evolutionary ecology studies have increasingly focused on the impact of intraspecific variability on population processes. However, the role such variation plays in the dynamics of spatially structured populations and how it interacts with environmental changes remains unclear. Here we experimentally quantify the relative importance of intraspecific variability in dispersal-related traits and spatiotemporal variability of environmental conditions for the dynamics of two-patch metapopulations using clonal genotypes of a ciliate in connected microcosms. We demonstrate that in our simple two-patch microcosms, differences among genotypes are at least as important as spatiotemporal variability of resources for metapopulation dynamics. Furthermore, we show that an important proportion of this effect results from variability of dispersal syndromes. These syndromes can therefore be as important for metapopulation dynamics as spatiotemporal variability of environmental conditions. This study demonstrates that intraspecific variability in dispersal syndromes can be key in the functioning of metapopulations facing environmental changes.

Diverse effects of nitric oxide reductase NorV on Aeromonas hydrophila virulence-associated traits under aerobic and anaerobic conditions.

NorV has been known to be an anaerobic nitric oxide reductase associated with nitric oxide (NO) detoxification. Recently, we showed that the norV gene of Aeromonas hydrophila was highly upregulated after co-culturing with Tetrahymena thermophila. Here, we demonstrated that the transcription and expression levels of norV were upregulated in a dose-dependent manner after exposure to NO under aerobic and anaerobic conditions. To investigate the roles of norV in resisting predatory protists and virulence of A. hydrophila, we constructed the norV gene-deletion mutant (ΔnorV). Compared to the wild type, the ΔnorV mutant showed no significant difference in growth at various NO concentrations under aerobic conditions but significantly stronger NO-mediated growth inhibition under anaerobic conditions. The deletion of norV exhibited markedly decreased cytotoxicity, hemolytic and protease activities under aerobic and anaerobic conditions. Also, the hemolysin co-regulated protein (Hcp) in the ΔnorV mutant showed increased secretion under aerobic conditions but decreased secretion under anaerobic conditions as compared to the wild-type. Moreover, the inactivation of norV led to reduced resistance to predation by T. thermophila, decreased survival within macrophages and highly attenuated virulence in zebrafish. Our data indicate a diverse role for norV in the expression of A. hydrophila virulence-associated traits that is not completely dependent on its function as a nitric oxide reductase. This study provides insights into an unexplored area of NorV, which will contribute to our understanding of bacterial pathogenesis and the development of new control strategies for A. hydrophila infection.

Gene identification and functional characterization of a Δ12 fatty acid desaturase in Tetrahymena thermophila and its influence in homeoviscous adaptation to low temperature.

Homeoviscous adaptation in poikilotherms is based in the regulation of the level of desaturation of fatty acids, variation in phospholipids head groups and sterol content in the membrane lipids, in order to maintain the membrane fluidity in response to changes in environmental temperature. Increased proportion of unsaturated fatty acids is thought to be the main response to low-temperature acclimation, which is mostly achieved by fatty acid desaturases. Genome analysis of the ciliate Tetrahymena thermophila and a gene knockout approach has allowed us to identify one Δ12 FAD and to study its activity in the original host and in a yeast heterologous expression system. The "PUFA index" -relative content of polyunsaturated fatty acids compared to the sum of saturated and monounsaturated fatty acid content- was ~57% lower at 15 °C and 35 °C in the Δ12 FAD gene knockout strain (KOΔ12) compared to WT strain. We characterized the role of T. thermophila Δ12 FAD on homeoviscous adaptation and analyzed its involvement in cellular growth, cold stress response, and membrane fluidity, as well as its expression pattern during temperature shifts. Although these alterations allowed normal growth in the KOΔ12 strain at 30 °C or higher temperatures, growth was impaired at temperatures of 20 °C or lower, where homeoviscous adaptation is impaired. These results stress the importance of Δ12 FAD in the regulation of cold adaptation processes, as well as the suitability of T. thermophila as a valuable model to investigate the regulation of membrane lipids and evolutionary conservation and divergence of the underlying mechanisms.

Homeorhesis and ecological succession quantified in synthetic microbial ecosystems.

The dynamics of ecological change following a major perturbation, known as succession, are influenced by random processes. Direct quantitation of the degree of contingency in succession requires chronological study of replicate ecosystems. We previously found that population dynamics in carefully controlled, replicated synthetic microbial ecosystems were strongly deterministic over several months. Here, we present simplified, two-species microbial ecosystems consisting of algae and ciliates, imaged in toto at single-cell resolution with fluorescence microscopy over a period of 1 to 2 weeks. To directly study succession in these ecosystems, we deliberately varied the initial cell abundances over replicates and quantified the ensuing dynamics. The distribution of abundance trajectories rapidly converged to a nearly deterministic path, with small fluctuations, despite variations in initial conditions, environmental perturbations, and intrinsic noise, indicative of homeorhesis. Homeorhesis was also observed for certain phenotypic variables, such as partitioning of the ciliates into distinct size classes and clumping of the algae. Although the mechanism of homeorhesis observed in these synthetic ecosystems remains to be elucidated, it is clear that it must emerge from the ways each species controls its own internal states, with respect to a diverse set of environmental conditions and ecological interactions.

Predator coevolution and prey trait variability determine species coexistence.

Predation is one of the key ecological mechanisms allowing species coexistence and influencing biological diversity. However, ecological processes are subject to contemporary evolutionary change, and the degree to which predation affects diversity ultimately depends on the interplay between evolution and ecology. Furthermore, ecological interactions that influence species coexistence can be altered by reciprocal coevolution especially in the case of antagonistic interactions such as predation or parasitism. Here we used an experimental evolution approach to test for the role of initial trait variation in the prey population and coevolutionary history of the predator in the ecological dynamics of a two-species bacterial community predated by a ciliate. We found that initial trait variation both at the bacterial and ciliate level enhanced species coexistence, and that subsequent trait evolutionary trajectories depended on the initial genetic diversity present in the population. Our findings provide further support to the notion that the ecology-centric view of diversity maintenance must be reinvestigated in light of recent findings in the field of eco-evolutionary dynamics.

Responses of Bioconvection of Tetrahymena thermophila to Partial Gravity Created by Aircraft Flight.

Bioconvection is a form of collective pattern formation driven by negative gravitaxis of swimming microorganisms. In bioconvection, the interaction between individual swimmers results in self-organization leading to the development of a macroscopic structure typically 100-1000 times greater than an individual microorganism. To gain insight into the role of gravity in this self-organization phenomenon, we investigated the bioconvective behavior of the ciliate Tetrahymena thermophila under short-term partial gravity, i.e., gravitational acceleration < 1 g, achieved by quasiparabolic flight maneuvers of an aircraft. The bioconvective responses of T. thermophila were assessed by observing the collective motion simultaneously in two separate scales, which we call macroscale and microscale, using a newly designed "dual-objective" device with two different magnifications. Microscale analysis revealed that the magnitude of gravikinesis, i.e., active regulation of the propulsive thrust, decreased almost linearly with changes in gravitational acceleration, while gravitactic characteristics, assessed by the distribution of the swimming direction, did not change significantly during partial gravity. Macroscale analysis demonstrated that downward plumes of convection pattern gradually shortened from the lower end, and disappeared under partial gravity. The sustained time of the plumes decreased almost linearly with changes in gravitational acceleration. The response of downward plumes to partial gravity may be attributable to the accumulation of cells into blobs in downward migration, which increases the rate of downward migration enough to exceed the rate of upward movement, which is enhanced due to gravikinesis. This suggests that gravity may act on cells involved in collective pattern formation differently than on free-swimming cells.

Selenium cytotoxicity in Tetrahymena thermophila: New clues about its biological effects and cellular resistance mechanisms.

Selenium is an essential micronutrient but at high concentrations can produce severe cytotoxicity and genomic damage. We have evaluated the cytotoxicity, ultrastructural and mitochondrial alterations of the two main selenium inorganic species; selenite and selenate, in the eukaryotic microorganism Tetrahymena thermophila. In this ciliate, selenite is more toxic than selenate. Their LC50 values were calculated as 27.65 µM for Se(IV) and 56.88 mM for Se(VI). Significant levels of peroxides/hydroperoxides are induced under low-moderate selenite or selenate concentrations. Se(VI) exposures induce an immediate mitochondrial membrane depolarization. Selenium treated cells show an intense vacuolization and some of them present numerous discrete and small electrondense particles, probably selenium deposits. Mitochondrial fusion, an intense swelling in peripheral mitochondria and mitophagy are detected in selenium treated cells, especially in those exposed to Se (IV). qRT-PCR analysis of diverse genes, encoding relevant antioxidant enzymes or other proteins, like metallothioneins, involved in an environmental general stress response, have shown that they may be crucial against Se(IV) and/or Se (VI) cytotoxicity.

A specialized condensin complex participates in somatic nuclear maturation in Tetrahymena thermophila.

Condensins are highly conserved proteins that are important for chromosome maintenance in nearly all forms of life. Although many organisms employ two forms of the condensin complex, the condensin genes in Tetrahymena have expanded even further. Here we report a form of condensin that is specifically active during sexual reproduction. This complex, condensin D, is composed of the core condensin proteins, Smc2 and Smc4, and two unique subunits, the kleisin Cph5 and Cpd2. Cpd2 is also found in somatic nuclei in vegetative cells, but is dispensable for growth and nuclear division. Immunoprecipitation experiments show that condensin D interacts with a putative member of a chromatin-remodeling complex during development. Condensin D is required for sexual reproduction and for endoreplication and genome reduction of the progeny's somatic nuclei. Altogether, Tetrahymena possesses at least four forms of condensin to fulfill the needs of maintaining chromosomes in two different nuclei containing the somatic and germline genomes.

Habitat choice meets thermal specialization: Competition with specialists may drive suboptimal habitat preferences in generalists.

Limited dispersal is classically considered as a prerequisite for ecological specialization to evolve, such that generalists are expected to show greater dispersal propensity compared with specialists. However, when individuals choose habitats that maximize their performance instead of dispersing randomly, theory predicts dispersal with habitat choice to evolve in specialists, while generalists should disperse more randomly. We tested whether habitat choice is associated with thermal niche specialization using microcosms of the ciliate Tetrahymena thermophila, a species that performs active dispersal. We found that thermal specialists preferred optimal habitats as predicted by theory, a link that should make specialists more likely to track suitable conditions under environmental changes than expected under the random dispersal assumption. Surprisingly, generalists also performed habitat choice but with a preference for suboptimal habitats. Since this result challenges current theory, we developed a metapopulation model to understand under which circumstances such a preference for suboptimal habitats should evolve. We showed that competition between generalists and specialists may favor a preference for niche margins in generalists under environmental variability. Our results demonstrate that the behavioral dimension of dispersal-here, habitat choice-fundamentally alters our predictions of how dispersal evolve with niche specialization, making dispersal behaviors crucial for ecological forecasting facing environmental changes.

Dual-stressor selection alters eco-evolutionary dynamics in experimental communities.

Recognizing when and how rapid evolution drives ecological change is fundamental for our understanding of almost all ecological and evolutionary processes such as community assembly, genetic diversification and the stability of communities and ecosystems. Generally, rapid evolutionary change is driven through selection on genetic variation and is affected by evolutionary constraints, such as tradeoffs and pleiotropic effects, all contributing to the overall rate of evolutionary change. Each of these processes can be influenced by the presence of multiple environmental stressors reducing a population's reproductive output. Potential consequences of multistressor selection for the occurrence and strength of the link from rapid evolution to ecological change are unclear. However, understanding these is necessary for predicting when rapid evolution might drive ecological change. Here we investigate how the presence of two stressors affects this link using experimental evolution with the bacterium Pseudomonas fluorescens and its predator Tetrahymena thermophila. We show that the combination of predation and sublethal antibiotic concentrations delays the evolution of anti-predator defence and antibiotic resistance compared with the presence of only one of the two stressors. Rapid defence evolution drives stabilization of the predator-prey dynamics but this link between evolution and ecology is weaker in the two-stressor environment, where defence evolution is slower, leading to less stable population dynamics. Tracking the molecular evolution of whole populations over time shows further that mutations in different genes are favoured under multistressor selection. Overall, we show that selection by multiple stressors can significantly alter eco-evolutionary dynamics and their predictability.

Restoration of cellular integrity following "ballistic" pronuclear exchange during Tetrahymena conjugation.

During sexual reproduction or conjugation, ciliates form a specialized cell adhesion zone for the purpose of exchanging gametic pronuclei. Hundreds of individual membrane fusion events transform the adhesion zone into a perforated membrane curtain, the mating junction. Pronuclei from each mating partner are propelled through this fenestrated membrane junction by a web of short, cris-crossing microtubules. Pronuclear passage results in the formation of two breaches in the membrane junction. Following pronuclear exchange and karyogamy (fertilization), cells seal these twin membrane breaches thereby re-establishing cellular independence. This would seem like a straightforward problem: simply grow membrane in from the edges of each breach in a fashion similar to how animal cells "grow" their cytokinetic furrows or how plant cells construct a cell wall during mitosis. Serial section electron microscopy and 3-D electron tomography reveal that the actual mechanism is less straightforward. Each of the two membrane breaches transforms into a bowed membrane assembly platform. The resulting membrane protrusions continue to grow into the cytoplasm of the mating partner, traverse the cytoplasm in anti-parallel directions and make contact with the plasma membrane that flanks the mating junction. This investigation reveals the details of a novel, developmentally-induced mechanism of membrane disruption and restoration associated with pronuclear exchange and fertilization in the ciliate, Tetrahymena thermophila.

Genome plasticity in response to stress in Tetrahymena thermophila: selective and reversible chromosome amplification and paralogous expansion of metallothionein genes.

Extreme stress situations can induce genetic variations including genome reorganization. In ciliates like Tetrahymena thermophila, the approximately 45-fold ploidy of the somatic macronucleus may enable adaptive responses that depend on genome plasticity. To identify potential genome-level adaptations related to metal toxicity, we isolated three Tetrahymena thermophila strains after an extended adaptation period to extreme metal concentrations (Cd2+ , Cu2+ or Pb2+ ). In the Cd-adapted strain, we found a approximately five-fold copy number increase of three genes located in the same macronuclear chromosome, including two metallothionein genes, MTT1 and MTT3. The apparent amplification of this macronuclear chromosome was reversible and reproducible, depending on the presence of environmental metal. We also analysed three knockout (KO) and/or knockdown (KD) strains for MTT1 and/or MTT5. In the MTT5KD strain, we found at least two new genes arising from paralogous expansion of MTT1, which encode truncated variants of MTT1. The expansion can be explained by a model based on somatic recombination between MTT1 genes on pairs of macronuclear chromosomes. At least two of the new paralogs are transcribed and upregulated in response to Cd2+ . Altogether, we have thus identified two distinct mechanisms, both involving genomic plasticity in the polyploid macronucleus that may represent adaptive responses to metal-related stress.

Biomechanics of Tetrahymena escaping from a dead end.

Understanding the behaviours of swimming microorganisms in various environments is important for understanding cell distribution and growth in nature and industry. However, cell behaviour in complex geometries is largely unknown. In this study, we used Tetrahymena thermophila as a model microorganism and experimentally investigated cell behaviour between two flat plates with a small angle. In this configuration, the geometry provided a 'dead end' line where the two flat plates made contact. The results showed that cells tended to escape from the dead end line more by hydrodynamics than by a biological reaction. In the case of hydrodynamic escape, the cell trajectories were symmetric as they swam to and from the dead end line. Near the dead end line, T. thermophila cells were compressed between the two flat plates while cilia kept beating with reduced frequency; those cells again showed symmetric trajectories, although the swimming velocity decreased. These behaviours were well reproduced by our computational model based on biomechanics. The mechanism of hydrodynamic escape can be understood in terms of the torque balance induced by lubrication flow. We therefore conclude that a cell's escape from the dead end was assisted by hydrodynamics. These findings pave the way for understanding cell behaviour and distribution in complex geometries.