Contingency and statistical laws in replicate microbial closed ecosystems.

Contingency, the persistent influence of past random events, pervades biology. To what extent, then, is each course of ecological or evolutionary dynamics unique, and to what extent are these dynamics subject to a common statistical structure? Addressing this question requires replicate measurements to search for emergent statistical laws. We establish a readily replicated microbial closed ecosystem (CES), sustaining its three species for years. We precisely measure the local population density of each species in many CES replicates, started from the same initial conditions and kept under constant light and temperature. The covariation among replicates of the three species densities acquires a stable structure, which could be decomposed into discrete eigenvectors, or "ecomodes." The largest ecomode dominates population density fluctuations around the replicate-average dynamics. These fluctuations follow simple power laws consistent with a geometric random walk. Thus, variability in ecological dynamics can be studied with CES replicates and described by simple statistical laws.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Evolutionary contribution to coexistence of competitors in microbial food webs.

The theory of species coexistence is a key concept in ecology that has received much attention. The role of rapid evolution for determining species coexistence is still poorly understood although evolutionary change on ecological time-scales has the potential to change almost any ecological process. The influence of evolution on coexistence can be especially pronounced in microbial communities where organisms often have large population sizes and short generation times. Previous work on coexistence has assumed that traits involved in resource use and species interactions are constant or change very slowly in terms of ecological time-scales. However, recent work suggests that these traits can evolve rapidly. Nevertheless, the importance of rapid evolution to coexistence has not been tested experimentally. Here, we show how rapid evolution alters the frequency of two bacterial competitors over time when grown together with specialist consumers (bacteriophages), a generalist consumer (protozoan) and all in combination. We find that consumers facilitate coexistence in a manner consistent with classic ecological theory. However, through disentangling the relative contributions of ecology (changes in consumer abundance) and evolution (changes in traits mediating species interactions) on the frequency of the two competitors over time, we find differences between the consumer types and combinations. Overall, our results indicate that the influence of evolution on species coexistence strongly depends on the traits and species interactions considered.

Evolution of density-dependent movement during experimental range expansions.

Range expansions and biological invasions are prime examples of transient processes that are likely impacted by rapid evolutionary changes. As a spatial process, range expansions are driven by dispersal and movement behaviour. Although it is widely accepted that dispersal and movement may be context-dependent, for instance density-dependent, and best represented by reaction norms, the evolution of density-dependent movement during range expansions has received little experimental attention. We therefore tested current theory predicting the evolution of increased movement at low densities at range margins using highly replicated and controlled range expansion experiments across multiple genotypes of the protist model system Tetrahymena thermophila. Although rare, we found evolutionary changes during range expansions even in the absence of initial standing genetic variation. Range expansions led to the evolution of negatively density-dependent movement at range margins. In addition, we report the evolution of increased intrastrain competitive ability and concurrently decreased population growth rates in range cores. Our findings highlight the importance of understanding movement and dispersal as evolving reaction norms and plastic life-history traits of central relevance for range expansions, biological invasions and the dynamics of spatially structured systems in general.

Raman Spectroscopic Characterization of Packaged L. pneumophila Strains Expelled by T. thermophila.

The intracellular lifestyle of L. pneumophila within protozoa is considered to be a fundamental process that supports its survival in nature. However, after ingesting the cells of L. pneumophila, some protozoa expel them as compressed live cells in the form of small rounded pellets. The pellets of tightly packaged viable but not culturable forms (VBNCFs) as well as highly infectious mature intracellular forms (MIFs) of L. pneumophila are considered as infectious particles most likely capable to cause human infection. Since L. pneumophila cells are hardly culturable from these pellets, detection methods for packaged live L. pneumophila forms remaining in water should be cultivation free. Hence, we demonstrate the potential of Raman microspectroscopy to directly sort pellets containing L. pneumophila cells, expelled by T. thermophila, and to characterize them on the basis of their Raman spectra.

Multicellular group formation in response to predators in the alga Chlorella vulgaris.

A key step in the evolution of multicellular organisms is the formation of cooperative multicellular groups. It has been suggested that predation pressure may promote multicellular group formation in some algae and bacteria, with cells forming groups to lower their chance of being eaten. We use the green alga Chlorella vulgaris and the protist Tetrahymena thermophila to test whether predation pressure can initiate the formation of colonies. We found that: (1) either predators or just predator exoproducts promote colony formation; (2) higher predator densities cause more colonies to form; and (3) colony formation in this system is facultative, with populations returning to being unicellular when the predation pressure is removed. These results provide empirical support for the hypothesis that predation pressure promotes multicellular group formation. The speed of the reversion of populations to unicellularity suggests that this response is due to phenotypic plasticity and not evolutionary change.

Selecting one of several mating types through gene segment joining and deletion in Tetrahymena thermophila.

The unicellular eukaryote Tetrahymena thermophila has seven mating types. Cells can mate only when they recognize cells of a different mating type as non-self. As a ciliate, Tetrahymena separates its germline and soma into two nuclei. During growth the somatic nucleus is responsible for all gene transcription while the germline nucleus remains silent. During mating, a new somatic nucleus is differentiated from a germline nucleus and mating type is decided by a stochastic process. We report here that the somatic mating type locus contains a pair of genes arranged head-to-head. Each gene encodes a mating type-specific segment and a transmembrane domain that is shared by all mating types. Somatic gene knockouts showed both genes are required for efficient non-self recognition and successful mating, as assessed by pair formation and progeny production. The germline mating type locus consists of a tandem array of incomplete gene pairs representing each potential mating type. During mating, a complete new gene pair is assembled at the somatic mating type locus; the incomplete genes of one gene pair are completed by joining to gene segments at each end of germline array. All other germline gene pairs are deleted in the process. These programmed DNA rearrangements make this a fascinating system of mating type determination.

Conjugation is necessary for a bacterial plasmid to survive under protozoan predation.

Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.

Membrane dynamics at the nuclear exchange junction during early mating (one to four hours) in the ciliate Tetrahymena thermophila.

Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila. Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membrane-bound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.