How does paramecium deal with environmental changes

Flavobacterium , Peptoniphilus , and Micrococcus , though not abundant, were only detected in the warmest treatment for the freshwater ciliate. In contrast, the DRB assemblage in E. The relative abundance of Vibrio sp. Ruficoccus -like species Verrucomicrobia were consistently present, accounting for a small portion 3. Nevertheless, warming induced a gradual decrease in its relative abundance.

Aestuariibacter and Pseudomonas spp. Figure 4. Variations in the assemblage structure of digestion-resistant bacteria DRB in association with two ciliates Paramecium bursaria and Euplotes vannus in different treatments. In different light environments, the composition of DRB in P. Structurally, Pseudoalteromonas spp. However, these two taxa were not detectable in the OTC treatments.

Pseudomonas in the control P. Figure 5. Note that Pseudomonas , Sulfitobacter , and Sphingomonas were highly antibiotic-resistant. Flectobacillus and Fluviicola were highly Pb-resistant. Hydrogenophaga was Hg-resistant, and both Aestuariibacter and Pelomonas were co-resistant to Pb and Cd. The bacteria associated with E.

In contrast to the non-detection of Sphingomonas in P. Both the Aestuariibacter and Ruficoccus -like phylotypes were abundant at the low-dose treatment but were reduced or disappeared at the higher doses Figure 5A. The DRB in the control P. There were many DRB persisting in the Hg treatments. The DRB assemblage in E. Furthermore, in the treatments of Cd0.

Ruficoccus -like Verrucomicrobia was minor 3. By using E. Overall, our molecular profiling of these two protist species indicated that experimentally manipulated warming, irradiance, antibiotic, and heavy metal stresses significantly influenced the structure of DRB assemblages. This provides evidence that the association between DRB and protists is highly dependent on environmental conditions. It was conceivable that there were consistently larger variations in the assemblage structure of DRB, relative to those in the environmental bacterioplankton across the treatments of E.

This is likely related to the fact that the variations in the environmental factors caused the shifts in the bacterioplankton community structure first Supplementary Table S1 , and then selective ingestion and differential digestion of the engulfed bacterial taxa took place next, which further screened out digestible populations, resulting in the altered assemblage structure of the bacteria i.

However, this reasoning cannot explain the results for P. The contrasting variability of DRB in P. The characteristic intracellular environment of mixotrophs thus selects a narrow spectrum of bacterial species to reside intracellularly. The selection of DRB in all the treatments investigated in this study may be related to the direct influence of environmental factors and pollution stresses on the physiology and biochemistry of the protists, which may affect the interactions between the predators and preys.

At a higher temperature not higher than the optimal temperature , a protist usually has a high growth rate Montagnes and Weisse, , ingestion rate Izaguirre et al.

Compared to the light condition, protozoan ingestion rate determined by using fluorescently labeled prey was lower under the dark conditions Izaguirre et al. Light availability is important for mixotrophs. For example, Chlorella symbionts provide photosynthetic products to their host under light conditions Shibata et al.

Heavy metals also affect protistan ingestion rates Al-Rasheid and Sleigh, and physiology Kim et al. The bacterial species associated with the protistan grazers may be related to not only the life stage and physiology of ciliates Xu et al.

Our analyses of clone libraries revealed that the overall DRB assemblage composition in E. The high proportions of Bacteroidetes and Betaproteobacteria could be due to their generally high abundance in freshwater systems Newton et al.

The phylum Bacteroidetes is well known for its high abundance in the gut microbiota Mahowald et al. It is likely that habitat freshwater-marine distinctness in higher taxonomic composition of DRB exists, but it is a notion that needs to be further investigated for more freshwater protist species. At lower taxonomic ranks, the DRB assemblage composition and structure along different environmental gradients and pollutant stresses showed many interesting characteristics, many of which are reported for the first time in this study, and the underlying mechanisms are discussed below.

It is interesting to observe that Pseudomonas spp. It was possible that the endosymbiotic green algae Chlorella sp. An opposite trend was observed for the dominant DRB group in the algae-free ciliate E. The cellular chemical composition of E. This temperature-driven stoichiometric shift in the host could select against specific heterotrophic DRB populations, such as Pseudoalteromonas spp.

The DRB assemblage significantly responded to the light conditions, which occurred only in the heterotrophic E. Interestingly, the DRB detected in the P. Typical species of the former two genera have also been demonstrated to be light-sensitive. Visible light can act to regulate a sensory module in Caulobacter crescentus in order to increase the biochemical activity and cellular signaling for cell—surface and cell—cell attachment Purcell et al.

Asticcacaulis was detected in the microbial consortium of the oil-rich green alga Botryococcus braunii Sambles et al. In the constant light treatment, continuous uptake of nutrients by the autotrophs, which included those in medium and the Chlorella symbionts inside the P. In constant darkness, P. Therefore, our study based on light manipulation and single cell analysis of protists provides evidence that mixotrophic protists are previously unrecognized hosts and micro-niches of these attaching bacteria in natural environments.

Cultivation in constant darkness or at night might lower the concentration of dissolved oxygen, which provides an ecological advantage to microaerobic bacteria, such as Curvibacter sp. Ding and Yokota, It has been demonstrated that members of this genus are able to inhibit fungal infection by interacting with other commensal bacterial species in the cnidarian Hydra Fraune et al.

In fact, at least filament fungi were recognizable via the naked eye at later periods of the cultures supplied with rice grains, indicating a risk of fungal infection of the P. In this sense, it is possible for these bacterial consortiums to function in anti-fungal activity.

This adaptive ability in such contrasting light environments is consistent with a previous study, which showed a combination of autotrophic and heterotrophic features in the genome of Variovorax paradoxus Han et al.

The DRB in the heterotrophic ciliate E. Alteromonas sp. A similar mechanism was proposed by a previous study Biller et al. The Pseudoalteromonas phylotypes were consistently present in the DRB assemblages under the conditions having a photic period, probably contributing to anti-fungal infection by more actively producing antifungal polyketide alteramides during the dark period Moree et al.

Thus, the co-occurrence of these two bacterial taxa may be of benefit to the host for a balance between rapid growth and low pathogenic infection under a natural diel light-dark condition Liu et al.

Gomiero and Viarengo showed that the antibiotic OTC 3. Such physiological shifts might have also occurred in these two species we investigated. Indeed, it has been demonstrated that many Pseudomonas species could degrade antibiotics via efflux pumps e.

Similarly, genes involved in aromatic compound catabolism and a type IV secretion system are present in the genome of a strain of Sulfitobacter Ankrah et al. Furthermore, Sulfitobacter strains isolated from marine hydrothermal vent fields were found to be antibiotic-resistant Farias et al.

All these suggest that the mixotrophic protist bearing endosymbiotic microalgae e. We detected these bacteria as a member of DRB in P. Furthermore, the Flectobacillus phylotypes became dominant in the Pb-treated cultures, demonstrating they are co-resistant to Pb and protistan digestion.

However, Flectobacillus spp. In the Hg-treated P. This suggests a strain-level differentiation in the heavy metal resistance of Flectobacillus.

Acinetobacter junii is able to form biofilms on surfaces to resistant to mercury Sarkar and Chakraborty, , which is in line with our observation of its survival in P. In addition, our results point to the non-digestible nature of this bacterial species in ciliated protozoa, highlighting its ecological success in natural environments. Furthermore, Hydrogenophaga was also abundant in P. It remains to be investigated how this hydrogen-oxidizing species interacts with the host and endosymbiotic green alga within the P.

Not much is known about the ecology and microbial association of Aestuariibacter , except for our recent report of this genus as a DRB in four marine and one freshwater species of ciliates Gong et al. In the present study, we once again found this genus as a dominant group of DRB in E. This is probably due to their resident habitats of high-pollution stresses, such as coastal seawater and tidal flats Yi et al.

Pelomonas was present in the DRB of E. Strains of Verrucomicrobia have been isolated as gut symbionts from sea cucumber, marine sponges, clamworm Wertz et al. Our study showed the association between a possible new genus of the family Puniceicoccaceae with the ciliate E.

As major players in the microbial loop and biogeochemical cycle within ecosystems, both bacteria and protists are ecologically linked not only through prey-predator food chains but also via collaboration in facing multiple stresses. Our study reveals for the first time that the assemblage composition and structure of ingested but inedible bacteria in two protists vary significantly in response to stresses of temperature, light, antibiotics, and heavy metals, indicating that the environment plays an important role in selecting specific bacterial populations in protist-bacteria associations.

Our results for these two ciliate species also provide an indication that trophic mode of protists mixotrophs vs. Furthermore, bacterial antibiotic resistance and metal resistance have been extensively studied in the past several decades.

The ecological interactions between these resistant bacteria and protists remain poorly understudied Nguyen et al. The datasets presented in this study can be found in online repositories. SZ did the data curation-equal, formal analysis-equal, investigation-equal, methodology-equal, resources-equal, software-equal, validation-equal, visualization-equal, and wrote the original draft-lead.

QZ performed the resources-supporting and validation-supporting. XZ and CD performed the validation-supporting. JG performed the conceptualization-lead, data curation-supporting, funding acquisition-lead, methodology-equal, project administration-lead, resources-equal, supervision-equal, validation-equal, and wrote, reviewed and edited the manuscript-lead.

All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Adams, H. Temperature controls on aquatic bacterial production and community dynamics in arctic lakes and streams. Effect of natural sunlight on bacterial activity and differential sensitivity of natural bacterioplankton groups in northwestern mediterranean coastal waters.

Al-Rasheid, K. The effects of heavy metals on the feeding rate of Euplotes mutabilis Tuffrau, Amin, S. Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria. Nature , 98— Ankrah, N. Draft genome sequence of Sulfitobacter sp. CB , a member of the Roseobacter clade of marine bacteria, isolated from an Emiliania huxleyi bloom.

Genome Announc. Ashelford, K. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras. Azam, F. The ecological role of water-column microbes in the sea. Bhat, S.

Effect of heavy metals on the performance and bacterial profiles of activated sludge in a semi-continuous reactor. Across this region, we identified 20 single-nucleotide polymorphisms that differed between clones 6.

We found no difference between subclones of the same clone. Sequencing the COI gene thus revealed the two clones to be genetically distinct at the sequence level in addition to their phenotypic differences. We tested the effects of osmotic and heat stress, alone and in combination, on infected and uninfected Paramecium.

Osmotic stress was achieved by adding 0. Salinity levels and temperatures are within the range of the ones for which Holospora has been shown to protect its host. The effect of these conditions was determined on a single Paramecium. For each combination of temperature, salinity, infection and genotype, we phenotyped 45 individuals. These 45 cells were spread over five blocks, each containing nine individuals from each treatment. The individuals from the two subclones of each genotype—infection combination were separate among blocks: one subclone seeded three blocks and the other seeded the two remaining blocks.

These blocks were used as the basic unit of replication in our statistical analyses. Each experimental block contained six plates comprising Paramecium. Paramecium clones, infected and uninfected individuals and salt exposure were arranged systematically across plates. Thus, for each experimental block, we calculated the proportion of survivors among the nine individuals per subclone and treatment.

We used factorial anova to analyse, first, how temperature and osmotic stress affected the survival of uninfected Paramecium from each clone. Subclone was nested within host clone and crossed with salt and temperature treatments. Proportion survival at the different time points was arcsine-square-root transformed to meet assumptions of anova. Where appropriate, initially, fully factorial statistical models were simplified by backward elimination of nonsignificant terms.

Second, we analysed the effects of the infection on the proportion survival in the presence or absence of abiotic stresses. Because of the high mortality under simultaneous salt and temperature stress, we did not have sufficient statistical resolution to analyse all four effects infection status, temperature treatment, salt treatment and host clone in a single anova.

Instead, we performed two separate analyses; one focussing on the temperature treatment and one on the salt treatment. Factorial statistical models were constructed by the same method as above, with the clonal replicate population nested within the host clone and infection status. All statistics were performed using the statistical software jmp version 5.

Both the high-salt and the high-temperature treatment reduced the survival of uninfected paramecia, but to different degrees Fig. Proportion of Paramecium caudatum surviving 3, 6, 9 and 24 h postexposure to osmotic and heat stress. This indicates, first, a synergistic effect of the two stresses: simultaneous exposure to high-salt and high-temperature treatments produced disproportionate rates of mortality Fig.

Note that the first divisions occurred between 9 and 24 h. Thus, there was no evidence for parasite-mediated heat-stress protection. At a high temperature, infection neither specifically increased nor decreased host survival and reproduction during the 24 h of the experiment.

Proportion of Paramecium caudatum surviving 24 h postexposure to heat stress: a clone K8 and b clone VEN. That is, the response to the salt treatment depended on both the infection status and the clone identity. For the VEN clone, infection decreased survival in the high-salt treatment at 6, 9 and 24 h postexposure; in contrast, for the K8 clone, infection increased survival in the high-salt treatment see multiple contrasts; Fig.

Thus, under osmotic stress, infection was costly for the VEN clone, while it was beneficial for the K8 clone. Despite this beneficial effect, both infected and uninfected VEN individuals had a higher general salt tolerance and thus higher survival than their K8 counterparts Fig.

Proportion of uninfected and infected Paramecium caudatum surviving 9 h postexposure to osmotic stress: a clone K8 and b clone VEN. The two P. We first discuss the combined effects of the two abiotic stressors in the absence of infection; second, we focus on the interactions between stress, infection and host genotype.

Our experiment confirms the negative effects of heat and osmotic stress on the survival of uninfected P. These results illustrate that it can be difficult for an organism to simultaneously manage two environmental stresses. Tolerance to heat stress in many organisms including Paramecium involves the upregulation of heat-shock proteins. It is unknown whether they function for tolerance against osmotic stress in P.

Osmoregulation in Paramecium relies on feedback between the internal mechanisms that control the osmolarity of the cell and permeability of the plasma membrane. Paramecium adapted to osmotic environments equal to or higher than, their intracellular environment will increase the ion concentration in their cells so that they can continue normal activity Stock et al.

However, there must be a limit beyond which they can no longer increase this concentration when water is drawn from the cell, causing it to rupture. Our results show that Paramecium cannot tolerate two abiotic stresses simultaneously.

Whether or not heat-shock proteins assist in tolerance to osmotic stress remains unclear. However, the high mortality under combined heat and osmotic stresses suggests that some of the tolerance mechanisms against these two stresses differ. A conflict may therefore arise between their simultaneous functioning, leading to a failure of either to function properly. Genetic variation between the two clones for tolerance against osmotic stress suggests that selection can act on this trait and that clone VEN would have a selective advantage over clone K8 under high-salt conditions.

This difference in salt tolerance could be attributable to a difference in the origin of the two clones and a difference in their evolutionary histories in environments with different salinities.

However, one should note that our two clones have spent several years in the lab before we conducted this assay. It is therefore difficult to attribute phenotypic differences to the evolutionary origins of each clone.

Previous studies revealed that Paramecium infected with Holospora spp. In this experiment, we found that infection provided protection against osmotic stress only, and this was true for only one host genotype. Contrary to reports with other Holospora species in other Paramecium genotypes, infection did not protect the host against heat stress. Nor did we find a synergistic effect of temperature and infection where the parasite becomes more virulent in more stressful environments, as observed frequently in other systems e.

Bedhomme et al. Infected individuals were observed to have lower levels of division, although this was not affected by temperature. The absence of an effect of infection on survival at different temperatures may be due to the host genotypes used in this experiment. Alternatively, it could also be that H. There was a difference between the two clones in their response to osmotic stress. Consistent with previous findings, we find evidence for parasite-mediated protection for clone K8, infected individuals showing higher survival.

The opposite was true for clone VEN, with infected individuals showing lower survival throughout the experiment, including under osmotic stress. One must be cautious in generalizing to species the observations based on one clone. Our results do not question the validity of the repeated observation that a Holospora symbiont can protect paramecia against abiotic stresses, but they do suggest that these effects may not be as universal as previously thought. There is then no reason why an already tolerant genotype should benefit from infection, being able to upregulate heat-shock proteins itself or already having high constitutive levels of hsps.

This may be true for the VEN clone in environments presenting an osmotic stress. Nonetheless, all infected paramecia must pay a cost to carrying the symbiont and providing the resources necessary for its growth. This cost may be offset, and thus hidden, when the host benefits from symbiont-induced tolerance to stress, but is visible when the host does not need the symbiont.

This cost may be reflected by the lower survival we observe for infected VEN individuals. The observation that different host genotypes do not benefit equally from infection relates to the standing genetic variation for tolerance against osmotic stress and the mechanisms by which stress adaptation may occur.

This observation has broad consequences for the evolution of host—parasite interactions towards mutualistic relationships. If a host already possesses tolerance to an abiotic stress, symbiont-mediated protection should provide no benefit. Consequently, an already tolerant genotype, such as for clone VEN, should not gain from infection and instead pays the cost of infection, as we observe.

Conversely, when a genotype benefits from symbiont-mediated protection in stressful environments, selection should favour infected individuals and the symbiont may become fixed in the population, possibly leading to mutualism. Weneed to be cautious, though, when interpreting these results. Selection would favour infected K8 hosts in a monoclonal population facing osmotic stress. However, this population would be easily invaded by both infected and uninfected VEN hosts, both having higher levels of survival than infected K8 hosts under osmotic stress.

This experiment reveals how different host genotypes respond to combinations of biotic and abiotic stresses. The lack of a general finding for parasite-mediated protection against abiotic stress for both genotypes highlights the need for multiple genotypes to be included in future investigation.

Further, we show how infection with a different, but closely related parasite, can differentially affect host life history.

We cannot be sure whether this result is attributable to host genotype or parasite species. When the contractile vacuole collapses, this excess water leaves the paramecium body through a pore in the pellicle "Biology of Paramecium". Perhaps the most unusual characteristic of paramecia is their nuclei. The two types of nuclei are the micronucleus and macronucleus.

The micronucleus is diploid ; that is, it contains two copies of each paramecium chromosome. Forney notes that the micronucleus contains all of the DNA that is present in the organism. On the other hand, the macronucleus contains a subset of DNA from the micronucleus, according to Forney. All Paramecium species have one macronucleus, according to Forney. However the number of micronuclei can vary by species. He gives the example of the Paramecium aurelia species complex, which have two micronuclei and Paramecium multimicronucleatum , which have several.

Why the presence of two distinct nuclei? One evolutionary reason is that it is a mechanism by which paramecia and other ciliates can stave off genetic intruders: pieces of DNA that embed themselves into the genome.

Paramecia can reproduce either asexually or sexually, depending on their environmental conditions. Asexual reproduction takes place when ample nutrients are available, while sexual reproduction takes place under conditions of starvation. During binary fission, one paramecium cell divides into two genetically identical offspring, or daughter cells. According to Forney, the micronucleus undergoes mitosis , but the macronucleus divides another way, called an amitotic, or non-mitotic, mechanism.

Conjugation among paramecia is akin to mating. Forney said that there are two mating types for paramecia, which are referred to as odd and even. This reflects the fact that the mating types for various Paramecium species are denoted by either an odd or even number.

For example, according to Forney, Paramecium tetraurelia have mating types 7 and 8. Moreover, only cells within a single Paramecium species can mate with one another. The process is easily distinguishable under laboratory conditions. They can actually form rather dramatic clumps of cells when they are initially mixed," Forney said.

During sexual reproduction, the micronuclei of each paramecium undergo meiosis , ultimately halving the genetic content to create a haploid nucleus. These are exchanged between the two connected mates. The haploid nuclei from each mate fuse to create a new, genetically varied, micronucleus.

During this process, the micronucleus replicates multiple times.