The locomotory and feeding responses of a Euplotes sp. As feeding began, the average path length shortened, the arcs became tighter, and the ciliates changed direction more frequently. The feeding activity of the Euplotes appeared to be gregarious, being concentrated in patches within the biofilm of attached bacteria. It was also noted that the feeding effort targeted patches previously visited by other Euplotes , despite reduced bacterial density relative to the surrounding field of attached bacteria. This focused and intense feeding activity resulted in localized zones of nearly complete clearance within the attached bacterial populations.
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Rheinheimera sp. In culture experiments, the number of ciliates treated both with liquid broth bacteria-free Supernatant treatment and bacteria plus medium Tq treatment , decreases with respect to control cells, with complete disappearance of ciliates within 6 h after Tq treatment. Results suggest that Rheinheimera sp. EpRS3 produces and releases in liquid culture one or more bioactive molecules affecting E.
TEM analysis of control not treated ciliates allowed to morphologically characterize both kind of E. In treated ciliates, collected soon after the arising of cell suffering leading to death, TEM observations revealed some ultrastructural damages, indicating that P.
Additionally, TEM investigation showed that when the ciliate culture was inoculated with Tq treatment, both a notable decrease of P. FISH experiments performed on treated ciliates confirmed TEM results and, by means of the specific probe herein designed, disclosed the presence of Rheinheimera sp. EpRS3 both inside phagosomes and free in cytoplasm in ciliates after Tq treatment.
This finding suggests a putative ability of Rheinheimera sp. EpRS3 to reintroduce itself in the environment avoiding ciliate digestion.
The strain was isolated from the rhizospheric soil of Echinacea purpurea Chiellini et al. Rheinheimera EpRS3 shows resistance to several antibiotic compounds Mengoni et al. Additionally, EpRS3 can inhibit the growth of opportunistic human pathogenic bacteria belonging to the Burkholderia cepacia complex Bcc Chiellini et al. It has been hypothesized that the production of antimicrobial compounds, and the subsequent antagonistic activity of some bacterial strains belonging to the same or different taxa, might play a key role in driving the structuring of microbial communities interacting with eukaryotic macro-organisms Maida et al.
The toxicity of bacteria belonging to Rheinheimera genus against other organisms has been also described in other strains such as GR5, showing antimicrobial activity against Gram-positive and Gram-negative bacteria, yeast, and algae Chlorophyceae, Trebouxiophyceae Chen et al.
Interestingly, Rheinheimera sp. Recently, the antibacterial toxicity of the marine bacterium Rheinheimera japonica KMM has been described as well. Moreover, the anti-quorum sensing activity of the diketopiperazine factor cyclo Trp-Ser produced by Rheinheimera aquimaris QSI02 against Chromobacterium violaceum CV and Pseudomonas aeruginosa PA01 has been evidenced, focusing the attention on the potentiality of this bacterial genus to interact with other bacterial species in environmental microbial communities Sun et al.
Under an ecological point of view, the production of bactericidal compounds from environmental bacterial strains can be related to different factors such as the competition for nutrients and the occupation of a specific niche by eliminating prior residents Hibbing et al. On the other side, bacteria in the environment can produce molecules affecting the survival of eukaryotic organisms such as fungi.
This is the case for example of some rhizospheric bacteria producing molecules with antifungal activity to protect the plant against pathogens e. Interestingly, few studies focused the attention on bacterial antimicrobial compounds affecting the growth and survival of protists. An example is that of the environmental pathogenic P. Lovejoy et al. A deleterious effect of freshwater bacteria Janthinobacterium lividum and C.
Chrysophyceae has also been described, by means of violacein production from bacterial strains Matz et al. In this scenario, a possible biocidal effect of Rheinheimera sp. EpRS3 against eukaryotic cells has become an intriguing topic. Could EpRS3 interact with eukaryotic unicellular microorganisms in complex microbial communities e.
Could this possible interaction determine the structuring of such communities in natural environments? To address these interesting questions, we performed experiments involving the ciliated protozoan Euplotes aediculatus Ciliophora, Spirotrichea monoclonal strain EASCc1, as a model organism to be tested in culture experiments with Rheinheimera sp.
The choice of E. Additionally, as symbioses involving ciliate hosts are widespread e. The symbiotic relationship between the endosymbiont P. The symbionts had assumed an obligate role: after their removal by ciliate ampicillin treatment, ciliates cannot properly divide anymore and after one week they eventually die Heckmann and Schmidt, EpRS3 on this ciliate, taken as a model system among freshwater ubiquitous grazing protozoans, in case the deleterious effect occurred in few hours [i.
EpRS3 on this ciliate, in case the deleterious effect occurred after about 1 day as a consequence of the depletion of the indispensable endosymbiont P. EpRS3 in case E. EpRS3 bioactive molecule s are shadowed by eukaryotic cells and do not affect host bacterial endosymbionts].
EpRS3 on Euplotes cells, with mitochondria being a probable target of the bioactive molecule s. Interestingly, TEM analysis on affected E. EpRS3 in Euplotes cytoplasm both inside and outside digestive vacuoles. A deleterious effect was also observed on the endosymbiont P.
Euplotes aediculatus specimens were collected from the anaerobic side tank 0 ppt salinity of the San Colombano activated sludge plant placed in Lastra a Signa Florence, Italy , in November A single cell was isolated from the original population and was washed several times in sterile San Benedetto water San Benedetto S. Italy in order to minimize the presence of contaminating microorganisms.
The cell was then accommodated in San Benedetto water and daily fed for 5 days with a drop of cerophyll medium CM inoculated with Raoultella planticola DSM Gammaproteobacteria , Enterobacteriaceae to induce its exponential growth.
CM is a commercially available mixture of pulverized wheat grasses. For the inoculation of CM, R. After discharging the supernatant, the pellet was washed and re-suspended with 1 ml of CM.
Once E. In this culture conditions, ciliate doubling time was about 36 h. However, all ciliates used in the experiments were kept in starving conditions for 4 days before the experiment to ensure food digestion. The culture was constantly checked to ensure cell healthy conditions during the whole time of the experiments. Living cells were immobilized on a slide for observation with the help of a coverslip without deforming them Skovorodkin, Ciliate identification was based on observations of morphological key-characters i.
Genomic DNA of Rheinheimera sp. Briefly, an isolated colony about 10 8 bacterial cells after 12 h growth, Lodish et al. The supernatant obtained after 3-min centrifugation at maximum speed was then placed in a sterile tube and used as template DNA for amplification.
A specific probe was designed in silico to detect Rheinheimera sp. FISH experiments were carried out according to the protocol by Manz et al. The experimental procedure has been resumed in Figure 1. After growing, the liquid culture was immediately processed; five different treatments were prepared to treat five separate 1-ml ciliate sub-cultures of E.
Previously, the ciliates were maintained in starving conditions for 4 days to let them complete digestion of their regular bacterial food Raoultella planticola and to minimize the presence of phagosomes with Gammaproteobacteria inside. All the sub-cultures received their specific treatment at the same time. Figure 1. Schematic representation of the procedure followed for the experimental design.
The first count was performed immediately after the beginning of each treatment. Then cell counts were performed every 60 min, until 24 h. When E. In order to study the possible cell damage that ciliates were facing, cell were fixed immediately before their death. A comparison with negative and positive control specimens was performed to highlight Euplotes cell structures affected by different EpRS3 treatments.
Any potentially observed treatment effect on the ciliate obligate endosymbiont P. A total of ten replicate experiments were conducted in different days in order to confirm the harmful effect of Rheinheimera sp. EpRS3 vs. Once the effect was confirmed, an experiment was conducted with the aim to obtain fixed cells treated and control ciliates for FISH and TEM analyses: three different culture replicates, monitored up to 24 h, were performed to test the effect of the five different treatments reported in Figure 1 on the growth and survival of the E.
Fluorescence in situ hybridization analysis was used both to assess the presence of the two different endosymbionts in E. EpRS3 inside E. About 15 ciliate cells were randomly collected from both experimental and control cultures and were washed three times in sterile San Benedetto water and separately fixed on slides for FISH experiments as described by Szokoli et al.
EpRS3 presence. Table 1. List of probes used for FISH experiments reporting probe sequence, their fluorochrome, specificity, formamide concentration, and application.
The pellet of Rheinheimera sp. Transmission electron microscopy analysis was used both to assess the morphology of the two different endosymbionts in E. After overnight growth in liquid TSB medium the pellet of Rheinheimera sp. Prokaryotic cells were fixed in 1. The production of hydrogen peroxide by Rheinheimera sp.
EpRS3 was assessed with a colorimetric assay on agar medium based on the Prussian blue-forming reaction Saito et al. The agar medium allows the identification of hydrogen peroxide-producing bacterial strains by the appearance of dark blue halos around the colonies, due to the formation of a precipitate. Ciliate strain EASCc1 was confirmed in morphological inspections as Euplotes aediculatus as perfectly matching the description of the species Curds, ; additionally, molecular analysis based on 18S rRNA gene sequencing confirmed the species assignation by morphological identification C.
Sigona, pers. The sequence obtained was bp long Acc. The Rheinheimera sp. The sequence was bp long Acc. Number MH, this work and However, several of the other treatments did affect ciliate survival Figure 2. Supernatant treatment showed conflicting results depending on the different replicate: in two out of ten replicates, cell number dramatically decreased between 6 and 24 h from T0 beginning of the treatment ; moreover, cells stopped moving 2—4 h from T0.
This behavior, with the same timing, was also recorded in Tq-treated ciliates.
Canadian Journal of Microbiology
We'd like to understand how you use our websites in order to improve them. Register your interest. Specimens of Euplotes were collected from Ross Sea Antarctica , allowed to multiply in the laboratory, and taxonomically studied on the basis of classical diagnostic traits as seen under the optical and scanning electron microscope. Two morphospecies were identified as new and named E. This is a preview of subscription content, log in to check access. Rent this article via DeepDyve.
The effect on protist grazing of diel variation of carbon to nitrogen ratio C:N in algal prey was investigated using the dinoflagellate Lepidodinium sp. Both predator and prey cultures were maintained in light:dark cycle, with an additional set of prey cultures in a reversed light:dark cycle to that of predator cultures. Grazing experiments were conducted near the end of light light experiment and dark dark experiment phase with the algal prey in the same and opposite phases provided as mono-diets. In all experiments, prey at the end of light phase day prey possessed higher C:N than prey at the end of dark phase night prey. Grazing rates in the light experiments were higher than in the dark experiments for both predators.
We'd like to understand how you use our websites in order to improve them. Register your interest. Pseudodiaptomus annandalei is an estuarine species and being cultured as live feed for grouper fish larvae and other planktivores. We examined the predation behavior of P.
Euplotes is a genus of ciliates in the subclass Euplotia. Species are widely distributed in marine and freshwater environments, as well as soil and moss. Most members of the genus are free-living, but two species have been recorded as commensal organisms in the digestive tracts of sea urchins. Euplotes cells are inflexible, dorsoventrally flattened, and roughly ovoid, with a very large oral region peristome bordered on the left by a long "adoral zone of membranelles " AZM. Like other spirotrich ciliates, Euplotes move and feed with the help of compound ciliary organelles called "cirri," made up of thick tufts of cilia sparsely distributed on the cell.