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Ciliates as model systems to study the effect of salinity stress on bacterial endosymbiont transmission

Chiara Pasqualetti
Giulio Petroi
Martina Schrallhammer
Saturday, November 21, 2015 to Thursday, December 31, 2015

Paramecium, unicellular ciliated eukaryotes, can be found in freshwater and brackish environments. Changes in salinity concentration present an environmental stress important for the ecology of these organisms. Additionally, they can harbor frequently endosymbionts. The resulting symbiosis between Paramecium and bacterial cells could be parasitic, mutualistic, or even commensalistic depending also on environmental conditions. About 60 different symbionts were observed to inhabit different cell compartments of Paramecium (Fokin & Görtz, 2009; Schweikert et al, 2013). In this work, the endosymbiosis between the ciliate host Paramecium and its symbiont Candidatus Megaira polyxenophila was analysed. Ca. M. polyxenophila belongs to the bacterial class of Alphaproteobacteria, order Rickettsiales, family Rickettsiaceae. All species of Rickettsiaceae share an obligate intracellular lifestyle (Lee et al., 2014; Schrallhammer et al., 2013).The aim of my project is to obtain genetically identical Paramecium lines, which are either infected or endosymbiont-free. Two different approaches were used to achieve this aim: an antibiotic treatment to cure infected paramecia and an infection experiment with naïve Paramecium lines. This is a prerequisite for subsequent functional analysis to expand our knowledge regarding the role of this endosymbiont in its host and in particular under salinity stress conditions. Three infected strains were used for the antibiotic treatment:

  • YE9 Paramecium biaurelia infected by Ca. M. polyxenophila in the cytoplasm
  • LgJac 2III Paramecium primaurelia infected by Ca. M. polyxenophila in the cytoplasm
  • Mue 186-50 Paramecium caudatum infected by Ca. M. polyxenophila in the macronucleus

Four endosymbiont-free strains of Paramecium biaurelia were used for the infection experiment:

  • YE4
  • Antibes
  • Rieff
  • Yamaguchi

The antibiotic treatment was performed as follows (modified from Krenek et al., 2011): approximately 30 cells of the stock culture were transferred to 500 µl of antibiotic solution and incubated for 24h at 19 °C. The cells were washed four times in sterile Volvic water. Single cells were incubated in antibiotic solution at 19 °C for another 24h or 48h. Then single Paramecium cells were either treated another time with the antibiotic for 24h or directly transferred to Cerophyl medium inoculated with Raoultella planticola as food organism.Following antibiotics in different concentrations were used:

  • Streptomycin (100 µg/ml, 150 µg/ml, 250 µg/ml, 300 µg/ml, 500 µg/ml)
  • Chloramphenicol (150 µg/ml)
  • Tetracycline (120 µg/ml, 130 µg/ml, 150 µg/ml)
  • Chloramphenicol + tetracycline (each 150 µg/ml)

The success of the antibiotic treatment was tested by fluorescence in situ hybridization (FISH). This method allows to visualize individual bacterial cells in situ the host cell. Of the tested antibiotics and concentrations, only with tetracycline in the range from 130 till 150 µg/ml a clear decrease of the intracellular bacteria and finally (after several rounds of treatment) a cure from Ca. M. polyxenophila was achieved. The antibiotic treatment was a challenging task and time consuming because Ca.Megaira, like other Rickettsiaceae (Rolain et al., 1998), is resistant to numerous antibiotics. In this it differs from other obligate Paramecium endosymbionts like for example Holospora and Caedibacter, which show both a much higher susceptibility regarding the used antibiotics (e.g. both can be easily eradicated using streptomycin) and the applied concentrations. Therefore, already existing protocols for Paramecium intracellular bacteria needed to be adapted to the special requirements of this endosymbiont.For the infection experiment, naïve strains of P. biaurelia were mixed with Ca. M. polyxenophila inoculum. This inoculum was obtained after lysis of infected Paramecium cells by mechanical shearing. Thus the intracellular bacteria were released to the medium and could be taken up via phagocytosis by the naïve paramecia. Before starting the experiment, the cell density of the donor and receiver strains were counted and adjusted to the same cell density. The exact determination of the used amounts of cells increases the reproducibility of the experiment. To increase the probability of infection success, the naïve strains were cultivated in standard medium with a gradually increased of the salinity concentration (up to 2 ‰). Elevated salinity stress is hypothesized to increase the frequency of horizontal transmission (Fokin & Sabanayeva, 1990).The infection experiment was not successful but the general approach using Ca. M. polyxenophila was established. Variation of some parameters like amount of infected cells used to prepare the inoculum, cultivation temperature, feeding frequency after infection, and identity of receiver strains should be tested and might lead to the expected result.During my STSM period I had the chance to learn new techniques like the antibiotic treatment, the infection experiment, and different types of cell fixation for fluorescence in situ hybridization. The performed studying period of 40 days was sufficient to obtain Paramecium strains genetically identical infected and endosymbiont-free via the antibiotic treatment, which will be used in fitness assays to determine the mutualistic, neutral, or parasitic effect of Ca. M. polyxenophila on it Paramecium hosts.

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