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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Infection ability of aposymbiotic P. bursaria by native and non-native strains of Chlorella
  5. Mixed infection experiment
  6. Experimental procedures
  7. Acknowledgements
  8. References

The ciliate Paramecium bursaria living in mutualistic relationship with the unicellular green alga Chlorella is known to be easily infected by various potential symbionts/parasites such as bacteria, yeasts and other algae. Permanent symbiosis, however, seems to be restricted to Chlorella taxa. To test the specificity of this association, we designed infection experiments with two aposymbiotic P. bursaria strains and Chlorella symbionts isolated from four Paramecium strains, seven other ciliate hosts and two Hydra strains, as well as three free-living Chlorella species. Paramecium bursaria established stable symbioses with all tested Chlorella symbionts of ciliates, but never with symbiotic Chlorella of Hydra viridissima or with free-living Chlorella. Furthermore, we tested the infection specificity of P. bursaria with a 1:1:1 mixture of three compatible Chlorella strains, including the native symbiont, and then identified the strain of the newly established symbiosis by sequencing the internal transcribed spacer region 1 of the 18S rRNA gene. The results indicated that P. bursaria established symbiosis with its native symbiont. We conclude that despite clear preferences for their native Chlorella, the host–symbiont relationship in P. bursaria is flexible.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Infection ability of aposymbiotic P. bursaria by native and non-native strains of Chlorella
  5. Mixed infection experiment
  6. Experimental procedures
  7. Acknowledgements
  8. References

Symbioses, in general, are defined as two or more species living together in beneficial coexistence (e.g. Margulis and Fester, 1992; Douglas, 1995). This type of mutualistic interaction plays an important role in maintaining populations living under precarious environmental conditions (Margulis and Fester, 1992; Dolan and Pérez, 2000). In symbiotic ciliates, potential phototrophic endosymbionts are ingested by the host, but are able to escape digestion (Görtz, 1996). Such mixotrophic organisms combine the advantages of a heterotrophic nutrition mode with autotrophic energy gain, either through sequestered chloroplasts (kleptoplasts) or through algal symbionts (Dolan, 1992; Jones, 1994). An example of the latter is one of the classical symbioses, the mutualistic relationship between the ciliate Paramecium bursaria (Hymenostomatia) and the unicellular green alga Chlorella (Trebouxiophyceae) (Karakashian, 1975; Reisser, 1986). This symbiosis seems to represent a permanent association with hereditary symbionts (Siegel, 1960), where each algal cell is enclosed in an individual host vacuole, i.e. the perialgal vacuole. The exclusive mutualistic relationship of P. bursaria with ‘zoochlorellae’ under natural conditions has long been considered as a fact, but natural populations of aposymbiotic P. bursaria have been recently reported (Tonooka and Watanabe, 2002).

Paramecium bursaria is known to be easily invaded by various potential symbionts/parasites such as bacteria, yeasts and algae (Görtz, 1982; Omura et al., 2004). Although an infection with different algal symbionts is possible (Weis, 1978; Schulze, 1951), permanent establishment of a stable symbiosis seems to be restricted to Chlorella. Molecular phylogenetic analyses of the endosymbionts of P. bursaria have identified several Chlorella strains involved (Huss et al., 1989; Nishihara et al., 1998; Hoshina et al., 2004; 2005). Different Chlorella species have been found to have distinct suitabilities for the establishment of stable symbioses in P. bursaria (Hirshon, 1969; Weis, 1978) and infection rates have been shown to be affected by specificity of host and potential symbiont, such as recognition of surface antigens, or by physiological conditions of the partners involved (Karakashian and Karakashian, 1973; Weis, 1979; Nishihara et al., 1998).

Despite numerous studies on the ciliate-Chlorella system, the mechanisms and constraints of infection and establishment of symbiosis are still not fully understood. For example, none of the reports on Chlorella symbioses has mentioned phylogenetic relatedness of compatible symbionts. In a previous study on the phylogeny of symbiotic Chlorella, we identified several strains of in hospite Chlorella (i.e. in the host) among different symbiotic ciliate species, but also within one single species from different habitats (Summerer et al., submitted).

In this study, we designed infection experiments with two aposymbiotic strains of P. bursaria to test (i) whether infection ability reflected phylogenetic relationships and (ii) whether the aposymbiotic host selected for its native Chlorella or not when offered two other compatible strains.

Infection ability of aposymbiotic P. bursaria by native and non-native strains of Chlorella

  1. Top of page
  2. Summary
  3. Introduction
  4. Infection ability of aposymbiotic P. bursaria by native and non-native strains of Chlorella
  5. Mixed infection experiment
  6. Experimental procedures
  7. Acknowledgements
  8. References

For these experiments, two aposymbiotic strains of P. bursaria (PbKM2w and PbPIBw) and 16 different Chlorella strains were used (Table 1). As symbiont density and contact time are known to play a role in successful infection (Weis and Ayala, 1979), we selected conditions favouring this process. Thus, after 7 days of coculturing aposymbiotic P. bursaria with dense Chlorella cultures (10 000 algae/Paramecium cell), we found 11 infective out of 16 Chlorella strains tested (Table 1). We considered successfully infected P. bursaria, those that did not show any differences in appearance (Fig. 1A and B) or behaviour compared with native strains and maintained a stable symbiosis for several months. Although all Chlorella strains were initially ingested and retained in food vacuoles (Fig. 1C), and sometimes also digested by P. bursaria, only the 11 infective Chlorella strains were able to establish a symbiosis in both ciliate strains.

Table 1.  Origin of the 16 Chlorella strains and summary of infection experiments with two aposymbiotic P. bursaria strains.
Algal strainNatural hostOriginInfectionaUncorrected p-distancebAccession numberc
PbPIBwPbKM2w
  • a.

    Infection success; (+), accepted as symbiont; (−), not accepted as symbiont.

  • b.

    Phylogenetic (uncorrected p-) distances of a 412-bp gene segment including the ITS-1 region of infected algal strains to P. bursaria PIB native symbionts (EF030582).

  • c.

    NCBI accession numbers of a 412-bp gene segment including the ITS-1 region as reported in Summerer and colleagues (submitted). Except for Hydra viridissima (Cnidaria) all other natural hosts are ciliates.

  • PbPIBw, aposymbiotic P. bursaria isolated from Piburger See, Austria; PbKM2w, aposymbiotic P. bursaria strain KM2 isolated in Japan.

Chlorella Stokesia PIBStokesia vernalisPiburger See, Austria++0.1984EF030572
Chlorella Pelagodileptus PIBPelagodileptus trachelioidesPiburger See, Austria++0.2040EF030573
Chlorella Stentor PIBStentor polymorphusPiburger See, Austria++0.2006EF030575
Chlorella Teuthophrys PIBTeuthophrys trisulca trisulcaPiburger See, Austria++0.2066EF030574
Chlorella Uroleptus PIBUroleptus sp.Piburger See, Austria++0.2017EF030571
Chlorella Askenasia PIBAskenasia chlorelligeraPiburger See, Austria++0.2012EF030576
Chlorella Askenasia GKSAskenasia chlorelligeraGossenköllesee, Austria++0.1978EF030577
Chlorella PbPIBParamecium bursariaPiburger See, Austria++EF030582
Chlorella PbWParamecium bursariaWildbichl, Austria++0EF030583
Chlorella OChParamecium bursariaKarelia, Russia++0EF030578
Chlorella PbKM2Paramecium bursariaShimane, Japan++0.1767EF030584
‘Chlorella’ Hydra KRANHydra viridissimaInnsbruck, Austriaxx
‘Chlorella’ Hydra multiheaded (MH)Hydra viridissimaFlorida, USAxx
‘Chlorella minutissima’ CCAP211/52free-livingCCALA/New Zealand0.2759EF030581
Chlorella sorokiniana Praha A14free-livingCCALA/Czech Republic0.2902EF030580
‘Chlorella’ saccharophila 211–1afree-livingSAG/Germany0.4610EF030587
image

Figure 1. Results of infection experiments of aposymbiotic P. bursaria showing the native symbiosis of P. bursaria KM2 (A) and examples of a successful (B) and an unsuccessful (C) infection after 7 days of coculturing. Note, that Chlorella is also ingested into food vacuoles in unsuccessful pairings. Scale bar: 10 μm.

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Both strains of P. bursaria were easily infected with their native Chlorella and with those of all other investigated P. bursaria strains, no matter whether they belonged to the ‘northern’ (i.e. Europe: PbPIB, PbW and OCh) or the ‘southern’ (i.e. Japan: PbKM2) ecotype (sensuKvitko et al., 2001). These results are in contrast to those of Weis (1978), who found Chlorella isolates of P. bursaria that did not infect aposymbiotic paramecia. However, these authors used different host and symbiont strains than the ones tested in this study. Moreover, we believe the much longer incubation times used in our study compared with only 20 h in Weis (1978) study, might also explain this discrepancy.

An infection with the symbionts of two Hydra viridissima strains failed in spite of multiple experiments with different cell densities, symbiont preparations and ex-symbiotic ‘storage’ times of Hydra symbionts (Table 1). Unsuccessful infection pairings also occurred with three free-living Chlorella taxa (Table 1). The ability of Chlorella to synthesize maltose and to tolerate acidic environments, as well as the possession of a specific recognition mechanism have been identified as prerequisites for establishing a symbiosis (Huss et al., 1993; Weis, 1979). Although we did not test those characteristics, Douglas and Huss (1986) and Huss and colleagues (1993) stated that successful endosymbionts only occur within lineages of algae which are predisposed for symbiosis. Thus, our finding of free-living Chlorella being incompatible for infection was not unexpected.

We used the sequences of the internal transcribed spacer region 1 (ITS-1) of the 18S rRNA gene of Chlorella strains reported by Summerer and colleagues (submitted) and calculated the phylogenetic distances using the software paup*4.0b6 (Swofford, 2000) to test whether there was a relationship to their infection ability. Compatible Chlorella from the same Paramecium ecotype (Kvitko et al., 2001) had identical DNA sequences, but those from the ‘southern’ ecotype (PbKM2) showed a sequence difference in the ITS-1 region of 17.67% (Table 1). Compatible Chlorella strains of other ciliate species had sequence divergences below 21% (19.78% to 20.66%). By contrast, the incompatible free-living taxa diverged by more than 27% in the same region (Table 1).

A close phylogenetic relationship of prospective symbionts could be a prerequisite for infection. It is likely that closely related algae have similar physiological properties and that P. bursaria and their symbionts have to be well adapted to each other interacting in multiple ways. An example of this tight coupling besides nutritional cooperations is that P. bursaria is able to synchronize the symbionts cell division to its own by regulating the cell cycle of endosymbiotic Chlorella and keeping the algal population constant until the host cell enters cytokinesis (Kadono et al., 2004). Nevertheless, we did neither have free-living Chlorella differing less than 27% nor find ciliate symbionts with more than 21% sequence divergence to conclude whether incompatibility was exclusively dependent on phylogenetic distance.

From the analysis of the scientific literature it was unclear, whether specific ciliate strains accept specific Chlorella symbionts or not. For example, Weis (1978) suggested that the syngenic identity of P. bursaria has nothing to do with their acceptance of specific Chlorella symbionts, whereas Hoshina and colleagues (2005) argued that the types of Chlorella symbionts depended on their Paramecium hosts' lineages. We showed that Chlorella isolated from four different P. bursaria strains were compatible to both host strains tested. These results indicate that besides from infectivity, additional factors play a role in the specific composition of the symbiosis. Specific adaptation of symbiotic partners to different environmental conditions, such as exposure to elevated solar UV radiation or temperature, could be another reason for finding distinct algal symbionts in natural ciliate populations (Kvitko et al., 2001; Sommaruga et al., 2006; Summerer et al., submitted). Thus, the actual availability of a necessarily compatible Chlorella symbiont in the respective environment seems to be important. Likewise, a shift in the composition of symbionts is known to occur in marine Symbiodinium (Dinophyceae) associations after environmental change and bleaching events (Baker, 2003).

One common feature observed in infection experiments of all investigated Chlorella strains was the uptake of several algal cells together into food vacuoles of P. bursaria in the course of minutes (Fig. 1). It is known that P. bursaria efficiently ingests algae other than their Chlorella symbionts (Hirshon, 1969; Karakashian and Karakashian, 1973). For example, in our case, P. bursaria was able to live in axenic conditions on the free-living strain Chlorella saccharophila for months (M. Summerer, pers. observation). A certain number of infective Chlorella strains escaped from the food vacuole and were found individually enclosed in perialgal vacuoles, mainly distributed at the periphery of the Paramecium cell (Fig. 1). This phenomenon has been observed in previous studies (e.g. Karakashian, 1975). However, the peripheral region of P. bursaria cells can also be infected by bacteria and yeasts, which are not considered to be beneficial symbionts (Görtz, 1982), so that the accumulation of ingested Chlorella near the cell surface seems to be a prerequisite for the establishment of a symbiosis and not its result (Omura et al., 2004). Moreover, Kodama and Fujishima (2005) recently demonstrated that potentially symbiotic algae were able to escape from the paramecia's food vacuole after the fusion of acidosoms and lysosoms and succeeded to establish endosymbiosis.

Mixed infection experiment

  1. Top of page
  2. Summary
  3. Introduction
  4. Infection ability of aposymbiotic P. bursaria by native and non-native strains of Chlorella
  5. Mixed infection experiment
  6. Experimental procedures
  7. Acknowledgements
  8. References

We also assessed the host–symbiont specificity in a mixed infection experiment using the native Chlorella strain of P. bursaria PbPIBw isolated from Piburger See, and two other compatible Chlorella strains including one from another hymenostome ciliate, Stokesia vernalis, isolated from the same lake, and Askenasia chlorelligera isolated from another lake (Gossenköllesee). These two non-native Chlorella strains had very similar phylogenetic distances to the native symbiont (Table 1).

The ITS-1 sequences of all three infection replicates turned out to be identical to each other and to the native Chlorella symbionts of P. bursaria PbPIB. Certainly, we cannot exclude the presence of a minimal number of actually different symbionts in our samples which potentially were not amplified by polymerase chain reaction (PCR). However, from a mixture of three compatible symbionts, P. bursaria at least largely preferred their native Chlorella to establish a symbiosis. Although previous studies on the symbiosis of P. bursaria have identified variations in the infection abilities of different algal symbionts (i.e. Schulze, 1951; Weis, 1978), our results are the first to evidence a highly sophisticated degree of specific symbiont recognition in P. bursaria. Likewise, Nishiguchi and colleagues (1998) found a pronounced dominance of the native symbionts in experimental colonization of the squid Euprymna scolopes with different luminous bacteria (Vibrio fisheri). Moreover, these authors also found a coherency between colonization competency of prospective symbionts and the phylogenetic relatedness of the partners.

In natural environments, species-specific symbioses with optimized adaptations of both partners, seem to be the rule in P. bursaria, however, the successful establishment of a mutualistic relationship with several Chlorella symbionts even from other ciliate species is possible. Our findings indicate that despite clear preferences for their native Chlorella, the host–symbiont relationship in P. bursaria seems to be flexible enough to adapt to environmental change by accepting non-native Chlorella at least under experimental conditions.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Infection ability of aposymbiotic P. bursaria by native and non-native strains of Chlorella
  5. Mixed infection experiment
  6. Experimental procedures
  7. Acknowledgements
  8. References

The ciliates and their symbionts were collected, identified and isolated as described elsewhere (Sonntag et al., 2006; Summerer et al., submitted). Chlorella-free (i.e. aposymbiotic) clones of P. bursaria were obtained by growing the symbiotic ciliates in permanent darkness for several weeks to months at 20°C on a bacterial diet supported by a lettuce culture medium enriched with 1.5% Chalkley's medium. The absence of symbiotic algae was regularly checked with an Olympus microscope (BX50).

Chlorella symbionts were grown in Woods Hole MBL medium (Guillard and Lorenzen, 1972), except for P. bursaria symbionts, which were cultured in Tamiya medium (Tamiya, 1957) or freshly isolated. The Chlorella of H. viridissima were freshly isolated from their hosts by mechanical disruption and centrifugation. These Chlorella did not grow ex-symbiotically, but survived long enough in liquid Woods Hole MBL medium to perform infection experiments. The aposymbiotic strains of ciliates and the Chlorella strains were grown in light/temperature controlled chambers under a 16:8 h light : dark photoperiod and at 17–20°C.

For the mixed infection experiment, the different Chlorella strains were concentrated by gentle centrifugation (8000 g, 5 min) and mixed in a 1:1:1 ratio (final density of Chlorella cells ∼106 ml−1) in Woods Hole MBL medium. Then, aposymbiotic P. bursaria PbPIBw (Chlorella : host ratio 10 000:1) were added for infection and after 24 h, not ingested algae were removed by filtration through a nylon net (20 μm mesh size). Then, the infected P. bursaria were cultured in sterile Woods Hole MBL medium enriched with 30 mg l−1 yeast extract, in 100-ml Schott glass bottles for 10 days. The infection experiment was performed in triplicates.

To identify which Chlorella strain was found in the newly established symbiosis, infected ciliates were filtered through a nylon net (20 μm mesh size) to remove free Chlorella cells. About 30 000 P. bursaria per infected culture (triplicates) were harvested by centrifugation (3000 g, 5 min) and the pellet was frozen in liquid nitrogen. Total DNA was extracted by a commercial DNA extraction kit (DNeasy® Plant Mini Kit/Quiagen) according to the manufacturer's directions, with the following modifications: 600 μl of precooled (0–4°C) P1-buffer and 20 μl of RnaseA (20 g l−1) were added to the frozen sample pellet and the mixture was sonicated on ice for 3 min at 42 W to ensure the rupture of the algal cell walls. After this step, we followed the standard extraction protocol. We amplified both strands of the ITS-1 using the following primers 5′-GGAGAAGTCGTAACAAGGTTTCCG-3′ and 5′-ATCCTGCAATTCACACCAAGTATCG-3′ (Huss et al., 2002). Polymerase chain reaction amplification was performed on a Techne PHC-3 Thermal Cycler. An initial denaturation step of 94°C for 5 min was followed by 35 cycles under the following conditions: 45 s denaturation at 94°C, 45 s annealing at 53°C and 60 s extension at 72°C, followed by a final extension step of 72°C for 5 min. To check the density of amplified DNA fragments, 2-μl aliquots of the PCR products were separated on a 1% TAE-agarose gel in TAE buffer and stained with ethidium bromide. The amplification products were purified with ExoSAP-IT®/USB-Corporation before direct sequencing (Sanger et al., 1977) with an ABI PRISM Big Dye Terminator Cycle Sequencing Kit using an ABI 3100 DNA sequencer, according to the manufacturer's instructions. The resulting 412-bp gene segments including the ITS-1 region were individually checked and aligned using the computer program ClustalX (Thompson et al., 1994).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Infection ability of aposymbiotic P. bursaria by native and non-native strains of Chlorella
  5. Mixed infection experiment
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work was supported by a grant from the Austrian Science Fund (FWF 16559-B06) to R.S. We thank I. Miwa, Ibaraki University, Japan, for the donation of P. bursaria KM2, K. Kvitko, University of St. Petersburg, Russia, for the donation of the symbiotic Chlorella strain Och, and B. Hobmayer and I. Phillipp, University of Innsbruck, Austria, for the donation of H. viridissima strains.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Infection ability of aposymbiotic P. bursaria by native and non-native strains of Chlorella
  5. Mixed infection experiment
  6. Experimental procedures
  7. Acknowledgements
  8. References
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