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High degree of specificity in the association between symbiotic betaproteobacteria and the host Euplotes (Ciliophora, Euplotia)
Available online at www.sciencedirect.com
ScienceDirect European Journal of Protistology 59 (2017) 124–132
High degree of specificity in the association between symbiotic betaproteobacteria and the host Euplotes (Ciliophora, Euplotia) Claudia Vanninia,∗ , Cristiana Sigonaa , Martin Hahnb , Giulio Petronia , Masahiro Fujishimac a
Department of Biology, University of Pisa, Pisa, 56126, Italy Research Institute for Limnology, University of Innsbruck, Mondsee, 5310, Austria c Department of Sciences, Biology Section, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi 753-8512, Japan b
Received 14 February 2017; received in revised form 3 April 2017; accepted 4 April 2017 Available online 13 April 2017
Abstract The Betaproteobacteria-Euplotes association is an obligatory symbiotic system involving a monophyletic group of ciliate species and two betaproteobacteria species which can be alternatively present. Recent data showed that this relationship has been established more than once and that several symbiont-substitution events took place, revealing a complex and intriguing evolutionary path. Due to the different evolutionary pathways followed by the different symbionts, each bacterial strain could have differentially evolved and/or lost functional traits. Therefore, we performed re-infection experiments, both by phagocytosis and by microinjection, to test the possible functional role of the different bacteria towards the ciliates. Our results confirm that the growth capacity of the host is indissolubly linked to the presence of its original symbionts. Results of the attempts of re-infection by phagocytosis showed that none of the bacteria is able to successfully colonize the host cytoplasm in this way, even if regularly ingested. Re-infection by microinjection succeed only in one case. Such results point to a high degree of specificity in the interactions between bacteria and Euplotes even after the invasion step. Due to a co-evolutive pathway of reciprocal adaptation, different degree of re-colonization ability could have been conserved by the different species and strains of the symbionts. © 2017 Elsevier GmbH. All rights reserved. Keywords: “Candidatus Protistobacter heckmanni”; Polynucleobacter; Polynucleobacter asymbioticus; Polynucleobacter necessarius; Protistobacter; Symbiosis
Introduction Symbiosis between bacteria and ciliated protists are a widespread phenomenon (Fokin 2012) and represent very good models for the study of several aspects of the interactions between prokaryotic and eukaryotic cells (Görtz 2006). Among them, the association between the protist ciliate ∗ Corresponding
author. E-mail address: [email protected] (C. Vannini).
http://dx.doi.org/10.1016/j.ejop.2017.04.003 0932-4739/© 2017 Elsevier GmbH. All rights reserved.
Euplotes and its betaproteobacterial endosymbionts is a very interesting system for investigations on microbial symbiosis establishment and evolution. A monophyletic group of Euplotes species permanently harbor these symbionts. The association is obligate for both partners, as, if deprived of their symbionts, the ciliates are no longer able to divide and die within two weeks, while the bacteria are not able to survive or grow outside their host (Heckmann 1975; Heckmann et al. 1983; Vannini et al. 2005, 2007a, 2012). Since the bacteria are essential for the host reproduction and survival, it
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has been hypothesized that they restored some basic function lost by the common ancestor of the host species (Heckmann et al. 1983). A series of trans-infection experiments showed that in some cases symbionts hosted by different Euplotes species and strains can replace each other and resume host cell division (Fujishima and Heckmann 1984; Heckmann 1980). Betaproteobacteria of the species Polynucleobacter necessarius or “Candidatus Protistobacter heckmanni” are alternatively present in the host cytoplasm apparently playing similar functional roles as obligate symbionts (Heckmann and Schmidt 1987; Vannini et al. 2012, 2013). While the genus “Candidatus Protistobacter” constitutes a phylogenetic cluster composed exclusively by these symbionts, the genus Polynucleobacter includes also free-living bacteria which are strictly related to the symbiotic P. necessarius (i.e. 16S rRNA gene sequence similarity even above 99%, Hahn et al. 2009), but never share the same endosymbiotic lifestyle (Vannini et al. 2007a, 2007b; Vannini et al. 2012). Phylogenetic investigations suggest that “Candidatus P. heckmanni” probably acquired the symbiotic life-style as a consequence of a single, unique evolutionary event (Vannini et al. 2012). On the contrary, the same data strongly indicate that in the past some free-living Polynucleobacter bacteria colonized independently several times cytoplasms of Euplotes spp. hosts, giving rise to several symbiotic P. necessarius lineages. Despite these recent investigations on the history of the symbiosis, nothing is still known about the function played by the symbionts. The comparison of a symbiotic and a freeliving Polynucleobacter genome cleared that the symbiont genome is largely a sub-set of the free-living one (Boscaro et al. 2013) and no-exotic genes, possibly encoding for particular “symbiotic” function, were found. On the other hand, free-living Polynucleobacter bacteria represent an ecologically and genomically quite diverse group of bacteria (Hahn et al. 2016a). This diversity within the genus Polynucleobacter, and especially within the subcluster PnecC to which both endosymbiotic and free-living species belong, reflects the ecological diversification, which took place among Polynucleobacter bacteria (Hahn et al., 2016a). It could be therefore possible that the free-living Polynucleobacter are somehow pre-adapted to become symbionts and to play an essential functional role for the ciliate survival. Moreover, due to the different evolutionary pathways followed by “Candidatus P. heckmanni” and the different lineages of P. necessarius, each bacterial strain could have differentially evolved and/or lost functional traits. Considering these findings, it is certainly of interest to test the eventual functional role of the different bacteria towards the host ciliates. For these reasons, we performed re-infection experiments using the two bacterial symbionts (“Candidatus P. heckmanni” and P. necessarius) and a free-living Polynucleobacter strain. Aims of the work are to verify (1) if the two symbiotic species can replace each other, (2) if the free-living Polynucleobacter strain permanently possesses the ability of colonizing the ciliate cytoplasm and (3) if free-living Polynucleobacter strains are able to substitute from a functional
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point of view the original symbiont and eventually restore the division capacity in the aposymbiotic host.
Material and Methods Ciliate and bacterial cultures Strain FSP1.4 of Euplotes harpa, hosting P. necessarius (Vannini et al., 2005), and strain POH1 of Euplotes woodruffi, hosting “Candidatus P. heckmanni” (Vannini et al. 2012, 2013), were used for trans-infection experiments. Ciliate cultures were maintained at 19 ◦ C in artificial seawater at 5‰ and weekly fed with a monoclonal culture of Dunaliella tertiolecta grown in the same medium. Polynucleobacter asymbioticus strain QLW-P1DMWA-1T (the type strain of the species representing free-living Polynucleobacter, sharing with the symbiont P. necessarius a 16S rRNA gene sequence similarity >99%, Hahn et al. 2016b) was maintained in liquid NSY medium as described elsewhere (Hahn et al. 2004).
Re-infection experiments Ciliates were deprived of their original symbionts by a treatment in Penicillin-G 1000 U/ml (in their culture medium) for 5 days. Subsequently, they were moved back to penicillinfree medium and re-infection attempts were performed by phagocytosis or by microinjection of the bacteria, as described below. Re-infection was performed using (I) the formerly present symbiont from the same host strain, (II) the other species of symbiont (i.e. the symbiont derived from the other ciliate species), (III) the free-living Polynucleobacter strain (QLW-P1DMWA-1T ) (Fig. 1). Ciliate ability of dividing was then verified by isolating single ciliate cells in glass wells and by counting the number of cells for 8 days (21 replicas for each experimental group) (Fig. 2). For each experiment, two control groups were also included: ciliates not deprived of their original symbionts and aposymbiotic ciliates (i.e. ciliates treated but not re-infected afterward). The efficiency of penicillin treatment as well as of reinfection attempts (i.e. presence/absence and identity of each kind of bacteria) was verified as described below. Overall, four experiments were run: (1) re-infection by phagocytosis with E. harpa (FSP1.4) as recipient cell, (2) re-infection by phagocytosis with E. woodruffi (POH1) as recipient cell, (3) re-infection by microinjection with E. harpa (FSP1.4) as recipient cell, (4) re-infection by microinjection with E. woodruffi (POH1) as recipient cell. Re-infection experiments by phagocytosis. 300 ml of donor ciliate culture was pre-treated with 0.2 mg/ml chloramphenicol for one night in order to remove bacterial contaminants from the culture. Such treatment proved to be effective in killing bacterial contaminants, without affecting the ciliate endosymbionts (Boscaro et al. 2013). The ciliates were then washed from chloramphenicol by harvest-
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C. Vannini et al. / European Journal of Protistology 59 (2017) 124–132 RECIPIENT
TRANSFERRED BACTERIA Polynucleobacter necessarius (symbiont from E. harpa FSP1.4)
Aposymbiotic Euplotes harpa FSP1.4
"Ca. Protistobacter heckmanni" (symbiont from E. woodruffi POH1)
Polynucleobacter asymbioticus (free-living strain QLW-P1DMWA-1T)
Polynucleobacter necessarius (symbiont from E. harpa FSP1.4)
Aposymbiotic Euplotes woodruffi POH1
"Ca. Protistobacter heckmanni" (symbiont from E. woodruffi POH1)
Polynucleobacter asymbioticus (free-living strain QLW-P1DMWA-1T)
Fig. 1. Re-infection experiments. The symbionts Polynucleobacter necessarius and “Candidatus Protistobacter heckmanni” and the free-living Polynucleobacter asymbioticus were transferred to apo-symbiotic Euplotes cells by feeding or microinjection. Each infection experiment was performed against 21 recipient cells. For growth-rate monitoring after re-infection attempts, controls (ciliates harboring their natural symbiont) and aposymbiotic controls (penicillin treated ciliates) were also included.
ing with centrifuge at 300 × g for 10 min and re-suspension in culture medium. Then, the cells were centrifuged again and suspended in 2 ml culture medium and homogenized by syringing for 20 min on ice. The homogenate was suspended in 50 ml of culture medium. 10 ml of the recipient ciliate culture (formerly treated with penicillin) were harvested by centrifuge and re-suspended in the donor ciliate homogenate. When the free-living Polynucleobacter strain QLW-P1DMWA-1T was used for infection, recipient ciliates were re-suspended in the diluted bacterial culture. After 48 h, 21 recipient cells were isolated for the growth check. Presence or absence and identity of eventual cytoplasmic bacteria inside recipient ciliates were checked both at the end of the penicillin treatment, at the end of the 48 h after mixing and at the end of the growth check (Fig. 2a). Re-infection experiments by microinjection. For microinjection, donor and recipient cells were suspended in a hanging drop 2% (v/v) solution of bovine serum albumin dissolved in a Dryl’s solution (Dryl 1959). The microinjection equipment and procedure were followed by Fujishima and Heckmann (1984). Approximately 10 pl of symbiontcontaining cytoplasm from the donor ciliates were injected into a recipient ciliate cell. In case of free-living Polynucleobacter strain (QLW-P1DMWA-1T ), the same volume of bacterial suspension was injected into the recipient cell. For each experimental group, 21 recipient ciliate cells were injected and each injected cell was cultivated for the following growth check. Presence or absence and identity of eventual cytoplasmic bacteria were performed on recipient ciliates both at the end of the penicillin treatment and at the end of the growth check (Fig. 2b).
Aceto-orcein staining In order to verify presence or absence of bacterial symbionts in the cytoplasm of the ciliate cells at the end of the antibiotic treatment, aceto-orcein staining was used. Ciliates were fixed with Carnoy’s fixative (Carnoy 1887) and stained with 0.5% (v/v) orcein dissolved in 100% acetic acid.
Fluorescence in situ hybridization (FISH) Fig. 2. Time-line of the performed experiments. Time and duration of each step of the experiments is shown, together with the techniques used for checking their effectiveness. (a) Re-infection by phagocytosis; (b) re-infection by microinjection. Pg: re-suspension of recipient ciliates in homogenate of donor ciliate or in free-living P. asymbioticus culture for phagocytosis; d: days.
For FISH experiments, ciliates were fixed with paraformaldehyde 4% (v/v). FISH was then performed as described by Manz et al. (1992). Probe PnecC-16S-445 (Hahn et al. 2005), specific for Polynucleobacter belonging to subcluster PnecC (Hahn 2003) and probe Proti 445 (Vannini et al. 2012), specific for “Candidatus P. heckmanni”, were used in order to discriminate between Polynucleobacter and “Candidatus Protistobacter” (Fig. 3).
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Fig. 3. The two studied symbiotic systems. Ciliate macronucleus is visible by DAPI staining in strain FSP1.4 of E. harpa (a) and in strain strain POH1 of E. woodruffi (b). Betaproteobacterial obligate symbionts are visible by Fluorescent In Situ Hybridization (FISH) with specific oligonucleotide probes: P. necessarius inside E. harpa (c and e) and “Ca. P. heckmanni” inside E. woodruffi (d and f). Bar: 10 m.
DNA extraction and 16S rRNA gene characterization As it is not possible to discriminate unambiguously between free-living and symbiotic Polynucleobacter by FISH, 16S rRNA gene characterization was performed to this purpose. Total genomic DNA was extracted by ciliates at the end of the re-infection experiment using the NucleoSpin Plant ® II (Macherey-Nagel ) following manufacturer instructions. Amplification was then performed with the annealing taking place at 50 ◦ C × 35 cycles or by a touchdown PCR (Don et al. 1991) with the annealing taking place at 63 ◦ C, 57 ◦ C and 50 ◦ C. As the number of ciliate cells remaining at the end of the experiments was frequently very low (a few tens or even less), the amount of starting DNA represented in some cases a limiting factor. In such cases, also semi-nested PCR were performed, in order to obtain a suitable amount of amplified
DNA for sequencing. PCR products were directly sequenced in order to obtain at least the first part of the 16S rRNA gene, where several single-nucleotide differences between the gene sequences of free-living and symbiotic Polynucleobacter are located. Primers used for PCR and for direct sequencing are reported elsewhere (Vannini et al. 2012). In one case, sequencing demonstrated that in one well (i.e. in one single Euplotes cell) the original symbiotic P. necessarius survived the penicillin treatment. This well was thus excluded from further analysis. The obtained sequence has been deposited in the European Nucleotide Archive database with the accession number LT718446.
Statistical analysis Ciliate cells which died within 24 h after the re-infection procedure were excluded, taking into account the possi-
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bility that some ciliates could have been damaged by the setting-up protocols (i.e. by microinjection). The ANalysis Of SIMilarity (ANOSIM) was used to evaluate the statistical significance of observed differences between groups of data using the software PAST (Hammer et al. 2001).
Results Antibiotic treatment The used antibiotic treatment was effective, as the absence of the symbiotic bacteria from the host cytoplasm at the end of the five-days period was confirmed by aceto-orcein observations. As the only exception, at the end of the counting period in the re-infection by phagocytosis experiment, where E. harpa was tentatively re-infected with the free-living Polynucleobacter, the presence of bacteria in the host cytoplasm of some ciliates was observed. The 16S rRNA gene characterization cleared that the original P. necessarius symbiont survived the antibiotic treatment. For this reason, this experiment was repeated a second time and only results from the repetition were used for further analysis and discussed.
Re-infection experiments by phagocytosis Re-infection experiments by phagocytosis with E. harpa as recipient cell. Control ciliates (symbiotic host cells) maintained their symbionts and regularly divided as expected during the counting period, reaching after 8 days an average number of cells per well of 12.5 ± 11.6. Expected results were also obtained for the aposymbiotic recipients treated with Penicillin-G, which did not divide (only one division in one well). They reached after 8 days an average number of cells per well of 0.3 ± 0.5, as some ciliates died during the last days of counting. E. harpa cells re-infected with their original P. necessarius ingested the mixed bacteria by phagocytosis: the presence of P. necessarius inside the phagocytotic vacuoles was demonstrated by FISH with the probe PnecC-16S-445 (Fig. 4) at 48 h after mixing with homogenate. Nevertheless, no P. necessarius were present neither in the vacuoles nor in the cytoplasm later, at the end of the growth check. At the end of the growth check, ciliates of this experimental group reached an average number of cells per well of 1.3 ± 0.9. E. harpa mixed with “Candidatus P. heckmanni” reached an average number of ciliate cells of 3.2 ± 2.1. In this case, no positive signal of the probe specific for the given symbiont was observed neither in the digestive vacuoles nor in the cytoplasm, both after the 48 h period in homogenate and after the 8 days of growth check. Similar results were obtained when E. harpa was mixed with the free-living Polynucleobacter asymbioticus. The average number of the recipients per well after 8 days was of 0.3 ± 0.5. The Anosim test gave a highly significant result (p < 0.01 and R = 0.39). The growth rate of
Fig. 4. Re-infection by phagocytosis. P. necessarius, labeled with the probe PnecC-16S-445, inside the phagocytotic vacuoles of reinfected E. harpa cells at 48 h after mixing with homogenate. Bar: 10 m.
ciliates throughout the entire counting period are reported in Fig. 5a. Re-infection experiments by phagocytosis with E. woodruffi as recipient cell. Control E. woodruffi ciliates (symbiotic host cells) maintained a high division rate, reaching an average number of cells of 38.7 ± 9.6 at day 8 of the counting period. On the contrary, average number of cells for aposymbiotic ciliates (treated cells not mixed with bacteria) was 0.3 ± 0.5. Re-infection attempt with the original symbiont (“Candidatus P. heckmanni”) by phagocytosis did not lead to a recovery of division rate, as the average number of ciliates per well at the end of the counting period was of 1.8 ± 1.4. Similar results were obtained for re-infection attempts performed with symbiotic and free-living Polynucleobacter (2.3 ± 2.2 and 2.1 ± 1.3 average cells per well, respectively). The presence of bacteria inside the ciliates was never detected by FISH, both after the 48 h period in homogenate and after the 8 days of growth check. The Anosim test gave a highly significant result (p < 0.01 and R = 0.60). The growth rate of recipient E. woodruffi are reported in Fig. 5c.
Re-infection experiments by microinjection Re-infection experiments by microinjection with E. harpa as recipient cell. Control and aposymbiotic E. harpa cil-
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Fig. 5. Growth rates of recipient ciliates. Number of ciliates counted during the eight-days counting period after the re-infection attempts. C: control ciliates; AC: aposymbiotic (penicillin-treated) ciliates; Pn: ciliates re-infected with P. necessarius; Cph: ciliates re-infected with “Ca. P. heckmanni”; Pa: ciliates re-infected with P. asymbioticus. (a and b) E. harpa as recipient ciliate; (c and d) E. woodruffi as recipient ciliate. (a and c) re-infection by phagoytosis; (b and d) re-infection by microinjection.
iates were represented by the same experimental groups tested for the previous experiments (see above). Recovery of division capacity was observed if E. harpa were injected with their original P. necessaries endosymbionts, as the average number of cells per well at the end of the 8 days counting period was of 29.1 ± 8.3. In this case, the presence of P. necessarius symbiont inside the cytoplasm was confirmed by the FISH experiments. On the contrary, re-infection attempts with “Candidatus P. heckmanni” and with the free-living Polynucleobacter strain did not result in a recovery of the fission ability (average number of ciliates per well at the end of the counting period was of 0.5 ± 0.5 and 0.6 ± 1.1, respectively). In these two experimental groups, the presence of bacteria inside the ciliates was never detected by FISH. The Anosim test gave a highly significant result (p < 0.01 and R = 0.50). The growth rates of the recipients are reported in Fig. 5b. Re-infection experiments by microinjection with E. woodruffi as recipient cell. Control and aposymbiotic E. woodruffi ciliates were represented by the same experimental groups tested for the re-infection by phagocytosis
experiments (see above). E. woodruffi cells injected with their original symbiont (“Candidatus P. heckmanni”) reached after the 8 days of the counting period an average number of ciliates per well of 0.9 ± 1.4. At the end of the experiment, the symbiotic bacteria were not observed inside the cytoplasm of the recipients by FISH. When re-infection by microinjection was performed with symbiotic P. necessarius, an average number of cells per well of 2.1 ± 1.8 was obtained and no bacteria were detected inside the re-infected ciliates at the end of the experiment. The same datum for re-infection with the free-living P. asymbioticus strain was of 0.6 ± 0.9 and FISH detection gave a negative result as well. The Anosim test gave a highly significant result (p < 0.01 and R = 0.63). The number of ciliates counted in the glass wells are shown in Fig. 5d.
Discussion Success of re-infection experiments was verified by checking the presence of the used bacterium inside the recipient
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ciliates and by monitoring the growth rate of the recipients. We chose to monitor the growth rate of the recipient cells, instead of just checking their ability to perform any division (even just one more), for two reasons: (1) when Euplotes hosting “Candidatus P. heckmanni” are deprived of their symbionts, they do not always lose immediately and completely the capacity of dividing (Vannini et al. 2012), (2) it has been shown that even Euplotes naturally hosting Polynucleobacter symbionts can be able to perform one or two divisions after being deprived of the symbionts (Vannini et al. 2007). Our results confirm anyway that the growth capacity of the Euplotes species harboring Polynucleobacter or “Candidatus P. heckmanni” is indissolubly linked to the presence of the symbionts. Indeed, whenever bacterial symbionts were absent, we never detected a growth rate resembling that of the control groups. Ciliates without bacteria are definitively not able to grow. Although all the experimental groups showed a high variability, this datum is clearly evident. In some of the experimental groups where a growth recovery was not achieved, a certain degree of mortality was also observed, still confirming previous results about the obligate nature of these symbioses. As the rapidity of the effect of the antibiotic treatment can be highly variable, a five-days Penicillin-G treatment was necessary in order to obtain an efficient removal of symbionts. The lack of success of at least some of the re-infection experiments could have been also influenced by the timing. Indeed, re-infection experiments were always performed immediately after the end of the treatment, but at present it is not possible to exclude that a critical time exists after which reinfection could be no longer possible. The development in the future of a quicker method for symbiont removal, not affecting the host health, could maybe allow to verify this hypothesis. Results of the attempts of re-infection by phagocytosis showed that, in the used conditions, none of the bacteria is able to successfully colonize the host in this way. Such result is different from the ones previously obtained by Heckmann in 1980, who successfully obtained re-colonization by phagocytosis after antibiotic treatment. In that case, due to the former unavailability of adequate techniques, no check at the end of the experiments was performed in order to verify the true identity of the bacteria inside the ciliates. On the basis of our experience, we believe that, possibly, in that case, the original symbionts were not always completely removed by penicillin treatment. As an alternative explanation, it should be taken into account that those previous experiments were performed using different combinations of bacterial and ciliate strains and this aspect could indeed affect the obtained results (see also below). In our tests, ingestion of the given bacteria was demonstrated at least in one case by FISH experiments, but no re-colonization of the host cytoplasm occurred, even when the ciliates were exposed to the their original symbionts. As it is hard to imagine a different way of entrance for the establishment of the association under natural condi-
tions, we can hypothesize that colonization by phagocytosis can succeed only if specific requirements are satisfied under contingent circumstances. First of all, only a few free-living Polynucleobacter (PnecC) species or genotypes could display the physiological traits required for switching to the symbiotic life-style. Indeed, it has been recently shown that ecophysiological and genomic traits of free-living Polynucleobacter strains can present conspicuous differences (Hahn et al. 2016a). Such variability could considerably lower the percentage of free-living Polynucleobacter which, at a certain time and in a certain place, can be able to colonize an Euplotes host entering by phagocytosis. For example, non-functional secretion system genes of horizontal origin have been found in the genome of a symbiotic Polynucleobacter (Boscaro et al. 2013), but they are not present in the genome of the free-living Polynucleobacter strain QLW-P1DMWA-1T (Hahn et al. 2012). Bacterial secretion system genes can be involved in the invasion process during the establishment of a symbiosis and are often found in a degraded form in symbiotic genomes (Dale and Moran 2006). These genes could have been acquired by horizontal transfer by a free-living Polynucleobacter strain and could have enabled it to colonize a ciliate host, then degenerating up to a loss of functionality. On the other side, it has been previously shown that co-ingestion with infectious bacteria can enhance the ability of free-living bacteria to colonize ciliated hosts (Fokin et al. 2004). Co-occurrence of Polynucleobacter free-living strains with highly infectious bacteria could also represent a mechanism mediating host invasion. Therefore, more than one contingent condition could actually determine the ability of a certain free-living Polynucleobacter strain in entering an Euplotes host and switching to the symbiotic life-style. Such hypotheses could explain from one side the observed present inability of the symbionts to re-colonize by phagocytosis even their original host, and, on the other side, the unsuccessful attempts of colonization by the free-living Polynucleobacter strain QLW-P1DMWA1T . In the natural environment, free-living Polynucleobacter (PnecC) bacteria are ubiquitous in freshwater systems with large populations and many different genotypes or species coexist in the same habitat (Jezberova et al. 2010; Jezbera et al. 2011). A high frequency of phagocytosis by freshwater ciliates is therefore absolutely plausible. This could greatly improve the probability for an Euplotes host to match sometimes a compatible Polynucleobacter strain under the right conditions for the establishment of a symbiotic association. In other words, while the wide range of possible matches between potential hosts and bacteria in the natural environment can occasionally lead to the successful establishment of new symbioses, it could be much harder to obtain the same result under experimental, standardized conditions. Re-infection attempts by microinjection allow to skip the first stages of invasion of the host cytoplasm by introducing the bacteria directly inside the host cell. Nevertheless, this kind of re-infection succeeded only in the case of the
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symbiotic Polynucleobacter re-introduced inside its original host (E. harpa). The microinjection technique did not allow a re-colonization of ciliates neither when cross-infection experiments were performed (i.e. infection of a different host by symbiotic bacteria: symbiotic Polynucleobacter injected in E. woodruffi and “Candidatus P. heckmanni” injected in E. harpa), nor when free-living Polynucleobacter were used. Such results point to a high degree of specificity in the interactions between each symbiont and its host even after the invasion step, largely limiting the possibility of obtaining artificially new host/bacteria symbiotic combinations. A coevolutive pathway of reciprocal adaptation in each specific symbiotic association could indeed explain the matter. On the other side, the failure of re-infection by microinjection of the symbiont “Candidatus P. heckmanni” inside its original host (E. woodruffi) is actually harder to explain. The easiest explanation could maybe reside in a higher degree of sensibility of this bacterium toward the microinjection technique. On the other side, the high complexity of the evolutive history of this association could maybe lead to a different hypothesis. The hypothesis that “Candidatus P. heckmanni” could represent the bacterium that first established the association with Euplotes has been proposed (Vannini et al. 2012). In addition, it has been demonstrated that the symbiosis with Polynucleobacter bacteria has been established repeatedly by different, independent evolutive events (Vannini et al. 2012). This means that the evolutionary path of “Candidatus P. heckmanni” as endosymbiont in Euplotes could have been longer with respect to that of Polynucleobacter symbionts. Moreover, different Polynucleobacter symbionts became endocytobionts at different times, displaying nowadays different degrees of coevolution with their specific host ciliate. Therefore, their re-colonization abilities could be widely diverse. This could also explain previous results obtain by Fujishima and Heckmann (1984) while performing trans-infection experiments by microinjection. They used several different strains and species of Euplotes with their obligate symbionts. Indeed, in that case, the cross-infection attempts succeeded only in a few cases, but not all. The more ancient evolutionary history of “Candidatus P. heckmanni” as symbiont could justify its complete inability of re-establish a stable association, even within its original host. Similarly, among the variety of Polynucleobacter symbiotic strains, different degree of re-colonization ability could have been conserved, thus explaining why we found the Polynucleobacter symbiont from E. harpa strain FSP1.4 able to re-colonize only its original host and only by microinjection procedure. Indeed, different degree of infection ability are shown even by highly infective symbiont of ciliates, like, for example, Holospora spp., when different combinations of host’s and symbiont’s strains or species are used (Fokin et al. 2005; Fujishima 2009; Fujishima and Fuijita 1985; Nidelet and Kaltz 2007). A wider set of data on symbiotic and freeliving bacterial strains, both from genomic and physiological approaches, could help to solve the matter in the future.
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Acknowledgements This work was supported by the European Commission FP7-PEOPLE-2009-IRSES project CINAR PATHOBACTER (247658, mobility support to CS), the University of Pisa (PRA 2016 58), and by the Japan Ministry of Education, Culture, Sports, Science and Technology, TOKUBETSUKEIHI, to MF. Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. S. Gabrielli and V. Boscaro are gratefully acknowledged, respectively, for help with graphic artwork and useful critical discussion of results.
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in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences. ISME J. 10, 1642–1655. Hahn, M.W., Lang, E., Brandt, U., Wu, Q.L., Scheuerl, T., 2009. Emended description of the genus Polynucleobacter and the species Polynucleobacter necessarius and proposal of two subspecies, P. necessarius subsp. necessarius subsp. nov. and P. necessarius subsp. asymbioticus subsp. nov. Int. J. Syst. Evol. Microbiol. 59, 2002–2009. Hahn, M.W., Poeckl, M., Wu, Q.L., 2005. Low intraspecific diversity in a Polynucleobacter subcluster population numerically dominating bacterioplankton of a freshwater pond. Appl. Environ. Microbiol. 71, 4539–4547. ˇ Hahn, M.W., Scheuerl, T., Jezberova, J., Koll, U., Jezbera, J., Simek, K., Vannini, C., Petroni, G., Wu, Q.L., 2012. The passive yet successful way of planktonic life: genomic and experimental analysis of the ecology of a free-living Polynucleobacter population. PLoS One 7, e32772. Hahn, M.W., Schmidt, J., Pitt, A., Taipale, S.J., Lang, E., 2016b. Reclassification of four Polynucleobacter necessarius strains as Polynucleobacter asymbioticus comb. nov., Polynucleobacter duraquae sp. nov., Polynucleobacter yangtzensis sp. nov., and Polynucleobacter sinensis sp. nov., and emended description of the species Polynucleobacter necessarius. Int. J. Syst. Evol. Microbiol. 66, 2883–2892. Hahn, M.W., Stadler, P., Wu, Q.L., Poeckl, M., 2004. The filtrationacclimatization method for isolation of an important fraction of the not readily cultivable bacteria. J. Microbiol. Methods 57, 379–390. Hammer, Ø., Harper, D.A.T., Ryan, P.D., 2001. PAST: paleontological statistics software package for education and data analysis. Palaeontol. Electron. 4, 9. Heckmann, K., 1975. Omikron, ein essentieller Endosymbiont von Euplotes aediculatus. J. Protozool. 22, 97–104. Heckmann, K., 1980. Omikron, an essential endosymbiont of Euplotes aediculatus. In: Schwemmler, W., Schenk, H.E.A. (Eds.), Endocytobiology. Walter de Gruyter & Co, Berlin, New York, pp. 393–400. Heckmann, K., Ten Hagen, R., Görtz, H.D., 1983. Freshwater Euplotes species with a 9 type 1 cirrus pattern depend upon endosymbionts. J. Protozool. 30, 284–289.
Heckmann, K., Schmidt, H.J., 1987. Polynucleobacter necessarius gen. nov. sp. nov., an obligately endosymbiotic bacterium living in the cytoplasm of Euplotes aediculatus. Int. J. Syst. Bacteriol. 37, 456–457. Jezbera, J., Jezberova, J., Brandt, U., Hahn, M.W., 2011. Ubiquity of Polynucleobacter necessarius subspecies asymbioticus results from ecological diversification. Environ. Microbiol. 13, 922–931. Jezbera, J., Jezberova, J., Brandt, U., Lindstrom, E.S., Langenheder, S., Hahn, M.W., 2010. Ubiquity of Polynucleobacter necessarius ssp. asymbioticus in lentic freshwater habitats of a heterogeneous 2000 km2 area. Environ. Microbiol 12, 658–669. Manz, W., Amann, R.I., Wagner, M., Schleifer, K.H., 1992. Phylogenetic oligodeoxynucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst. Appl. Microbiol. 15, 593–600. Nidelet, T., Kaltz, O., 2007. Direct and correlated responses to selection in a host-parasite system: testing for the emergence of genotype specificity. Evolution 61, 1803–1811. Vannini, C., Ferrantini, F., Ristori, A., Verni, F., Petroni, G., 2012. Betaproteobacterial symbionts of the ciliate Euplotes: origin and tangled evolutionary path of an obligate microbial association. Environ. Microbiol. 14, 2553–2563. Vannini, C., Ferrantini, F., Verni, F., Petroni, G., 2013. A new obligate bacterial symbiont colonizing the ciliate Euplotes in brackish and freshwater: ‘Candidatus Protistobacter heckmanni’. Aquat. Microb. Ecol. 70, 233–243. Vannini, C., Lucchesi, S., Rosati, G., 2007a. Polynucleobacter: symbiotic bacteria in ciliates compensate for a genetic disorder in glycogenolysis. Symbiosis 44, 85–91. Vannini, C., Petroni, G., Verni, F., Rosati, G., 2005. Polynucleobacter bacteria in the brackish-water species Euplotes harpa (Ciliata Hypotrichia). J. Eukaryot. Microbiol. 52, 116–122. Vannini, C., Poeckl, M., Petroni, G., Wu, Q.L., Lang, E., Stackebrandt, E., Schrallhammer, M., Richardson, P.M., Hahn, M.W., 2007b. Endosymbiosis in statu nascendi: close phylogenetic relationship between obligately endosymbiotic and obligately free-living Polynucleobacter strains (Betaproteobacteria). Environ. Microbiol. 9, 347–359.
ScienceDirect European Journal of Protistology 59 (2017) 124–132
High degree of specificity in the association between symbiotic betaproteobacteria and the host Euplotes (Ciliophora, Euplotia) Claudia Vanninia,∗ , Cristiana Sigonaa , Martin Hahnb , Giulio Petronia , Masahiro Fujishimac a
Department of Biology, University of Pisa, Pisa, 56126, Italy Research Institute for Limnology, University of Innsbruck, Mondsee, 5310, Austria c Department of Sciences, Biology Section, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi 753-8512, Japan b
Received 14 February 2017; received in revised form 3 April 2017; accepted 4 April 2017 Available online 13 April 2017
Abstract The Betaproteobacteria-Euplotes association is an obligatory symbiotic system involving a monophyletic group of ciliate species and two betaproteobacteria species which can be alternatively present. Recent data showed that this relationship has been established more than once and that several symbiont-substitution events took place, revealing a complex and intriguing evolutionary path. Due to the different evolutionary pathways followed by the different symbionts, each bacterial strain could have differentially evolved and/or lost functional traits. Therefore, we performed re-infection experiments, both by phagocytosis and by microinjection, to test the possible functional role of the different bacteria towards the ciliates. Our results confirm that the growth capacity of the host is indissolubly linked to the presence of its original symbionts. Results of the attempts of re-infection by phagocytosis showed that none of the bacteria is able to successfully colonize the host cytoplasm in this way, even if regularly ingested. Re-infection by microinjection succeed only in one case. Such results point to a high degree of specificity in the interactions between bacteria and Euplotes even after the invasion step. Due to a co-evolutive pathway of reciprocal adaptation, different degree of re-colonization ability could have been conserved by the different species and strains of the symbionts. © 2017 Elsevier GmbH. All rights reserved. Keywords: “Candidatus Protistobacter heckmanni”; Polynucleobacter; Polynucleobacter asymbioticus; Polynucleobacter necessarius; Protistobacter; Symbiosis
Introduction Symbiosis between bacteria and ciliated protists are a widespread phenomenon (Fokin 2012) and represent very good models for the study of several aspects of the interactions between prokaryotic and eukaryotic cells (Görtz 2006). Among them, the association between the protist ciliate ∗ Corresponding
author. E-mail address: [email protected] (C. Vannini).
http://dx.doi.org/10.1016/j.ejop.2017.04.003 0932-4739/© 2017 Elsevier GmbH. All rights reserved.
Euplotes and its betaproteobacterial endosymbionts is a very interesting system for investigations on microbial symbiosis establishment and evolution. A monophyletic group of Euplotes species permanently harbor these symbionts. The association is obligate for both partners, as, if deprived of their symbionts, the ciliates are no longer able to divide and die within two weeks, while the bacteria are not able to survive or grow outside their host (Heckmann 1975; Heckmann et al. 1983; Vannini et al. 2005, 2007a, 2012). Since the bacteria are essential for the host reproduction and survival, it
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has been hypothesized that they restored some basic function lost by the common ancestor of the host species (Heckmann et al. 1983). A series of trans-infection experiments showed that in some cases symbionts hosted by different Euplotes species and strains can replace each other and resume host cell division (Fujishima and Heckmann 1984; Heckmann 1980). Betaproteobacteria of the species Polynucleobacter necessarius or “Candidatus Protistobacter heckmanni” are alternatively present in the host cytoplasm apparently playing similar functional roles as obligate symbionts (Heckmann and Schmidt 1987; Vannini et al. 2012, 2013). While the genus “Candidatus Protistobacter” constitutes a phylogenetic cluster composed exclusively by these symbionts, the genus Polynucleobacter includes also free-living bacteria which are strictly related to the symbiotic P. necessarius (i.e. 16S rRNA gene sequence similarity even above 99%, Hahn et al. 2009), but never share the same endosymbiotic lifestyle (Vannini et al. 2007a, 2007b; Vannini et al. 2012). Phylogenetic investigations suggest that “Candidatus P. heckmanni” probably acquired the symbiotic life-style as a consequence of a single, unique evolutionary event (Vannini et al. 2012). On the contrary, the same data strongly indicate that in the past some free-living Polynucleobacter bacteria colonized independently several times cytoplasms of Euplotes spp. hosts, giving rise to several symbiotic P. necessarius lineages. Despite these recent investigations on the history of the symbiosis, nothing is still known about the function played by the symbionts. The comparison of a symbiotic and a freeliving Polynucleobacter genome cleared that the symbiont genome is largely a sub-set of the free-living one (Boscaro et al. 2013) and no-exotic genes, possibly encoding for particular “symbiotic” function, were found. On the other hand, free-living Polynucleobacter bacteria represent an ecologically and genomically quite diverse group of bacteria (Hahn et al. 2016a). This diversity within the genus Polynucleobacter, and especially within the subcluster PnecC to which both endosymbiotic and free-living species belong, reflects the ecological diversification, which took place among Polynucleobacter bacteria (Hahn et al., 2016a). It could be therefore possible that the free-living Polynucleobacter are somehow pre-adapted to become symbionts and to play an essential functional role for the ciliate survival. Moreover, due to the different evolutionary pathways followed by “Candidatus P. heckmanni” and the different lineages of P. necessarius, each bacterial strain could have differentially evolved and/or lost functional traits. Considering these findings, it is certainly of interest to test the eventual functional role of the different bacteria towards the host ciliates. For these reasons, we performed re-infection experiments using the two bacterial symbionts (“Candidatus P. heckmanni” and P. necessarius) and a free-living Polynucleobacter strain. Aims of the work are to verify (1) if the two symbiotic species can replace each other, (2) if the free-living Polynucleobacter strain permanently possesses the ability of colonizing the ciliate cytoplasm and (3) if free-living Polynucleobacter strains are able to substitute from a functional
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point of view the original symbiont and eventually restore the division capacity in the aposymbiotic host.
Material and Methods Ciliate and bacterial cultures Strain FSP1.4 of Euplotes harpa, hosting P. necessarius (Vannini et al., 2005), and strain POH1 of Euplotes woodruffi, hosting “Candidatus P. heckmanni” (Vannini et al. 2012, 2013), were used for trans-infection experiments. Ciliate cultures were maintained at 19 ◦ C in artificial seawater at 5‰ and weekly fed with a monoclonal culture of Dunaliella tertiolecta grown in the same medium. Polynucleobacter asymbioticus strain QLW-P1DMWA-1T (the type strain of the species representing free-living Polynucleobacter, sharing with the symbiont P. necessarius a 16S rRNA gene sequence similarity >99%, Hahn et al. 2016b) was maintained in liquid NSY medium as described elsewhere (Hahn et al. 2004).
Re-infection experiments Ciliates were deprived of their original symbionts by a treatment in Penicillin-G 1000 U/ml (in their culture medium) for 5 days. Subsequently, they were moved back to penicillinfree medium and re-infection attempts were performed by phagocytosis or by microinjection of the bacteria, as described below. Re-infection was performed using (I) the formerly present symbiont from the same host strain, (II) the other species of symbiont (i.e. the symbiont derived from the other ciliate species), (III) the free-living Polynucleobacter strain (QLW-P1DMWA-1T ) (Fig. 1). Ciliate ability of dividing was then verified by isolating single ciliate cells in glass wells and by counting the number of cells for 8 days (21 replicas for each experimental group) (Fig. 2). For each experiment, two control groups were also included: ciliates not deprived of their original symbionts and aposymbiotic ciliates (i.e. ciliates treated but not re-infected afterward). The efficiency of penicillin treatment as well as of reinfection attempts (i.e. presence/absence and identity of each kind of bacteria) was verified as described below. Overall, four experiments were run: (1) re-infection by phagocytosis with E. harpa (FSP1.4) as recipient cell, (2) re-infection by phagocytosis with E. woodruffi (POH1) as recipient cell, (3) re-infection by microinjection with E. harpa (FSP1.4) as recipient cell, (4) re-infection by microinjection with E. woodruffi (POH1) as recipient cell. Re-infection experiments by phagocytosis. 300 ml of donor ciliate culture was pre-treated with 0.2 mg/ml chloramphenicol for one night in order to remove bacterial contaminants from the culture. Such treatment proved to be effective in killing bacterial contaminants, without affecting the ciliate endosymbionts (Boscaro et al. 2013). The ciliates were then washed from chloramphenicol by harvest-
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C. Vannini et al. / European Journal of Protistology 59 (2017) 124–132 RECIPIENT
TRANSFERRED BACTERIA Polynucleobacter necessarius (symbiont from E. harpa FSP1.4)
Aposymbiotic Euplotes harpa FSP1.4
"Ca. Protistobacter heckmanni" (symbiont from E. woodruffi POH1)
Polynucleobacter asymbioticus (free-living strain QLW-P1DMWA-1T)
Polynucleobacter necessarius (symbiont from E. harpa FSP1.4)
Aposymbiotic Euplotes woodruffi POH1
"Ca. Protistobacter heckmanni" (symbiont from E. woodruffi POH1)
Polynucleobacter asymbioticus (free-living strain QLW-P1DMWA-1T)
Fig. 1. Re-infection experiments. The symbionts Polynucleobacter necessarius and “Candidatus Protistobacter heckmanni” and the free-living Polynucleobacter asymbioticus were transferred to apo-symbiotic Euplotes cells by feeding or microinjection. Each infection experiment was performed against 21 recipient cells. For growth-rate monitoring after re-infection attempts, controls (ciliates harboring their natural symbiont) and aposymbiotic controls (penicillin treated ciliates) were also included.
ing with centrifuge at 300 × g for 10 min and re-suspension in culture medium. Then, the cells were centrifuged again and suspended in 2 ml culture medium and homogenized by syringing for 20 min on ice. The homogenate was suspended in 50 ml of culture medium. 10 ml of the recipient ciliate culture (formerly treated with penicillin) were harvested by centrifuge and re-suspended in the donor ciliate homogenate. When the free-living Polynucleobacter strain QLW-P1DMWA-1T was used for infection, recipient ciliates were re-suspended in the diluted bacterial culture. After 48 h, 21 recipient cells were isolated for the growth check. Presence or absence and identity of eventual cytoplasmic bacteria inside recipient ciliates were checked both at the end of the penicillin treatment, at the end of the 48 h after mixing and at the end of the growth check (Fig. 2a). Re-infection experiments by microinjection. For microinjection, donor and recipient cells were suspended in a hanging drop 2% (v/v) solution of bovine serum albumin dissolved in a Dryl’s solution (Dryl 1959). The microinjection equipment and procedure were followed by Fujishima and Heckmann (1984). Approximately 10 pl of symbiontcontaining cytoplasm from the donor ciliates were injected into a recipient ciliate cell. In case of free-living Polynucleobacter strain (QLW-P1DMWA-1T ), the same volume of bacterial suspension was injected into the recipient cell. For each experimental group, 21 recipient ciliate cells were injected and each injected cell was cultivated for the following growth check. Presence or absence and identity of eventual cytoplasmic bacteria were performed on recipient ciliates both at the end of the penicillin treatment and at the end of the growth check (Fig. 2b).
Aceto-orcein staining In order to verify presence or absence of bacterial symbionts in the cytoplasm of the ciliate cells at the end of the antibiotic treatment, aceto-orcein staining was used. Ciliates were fixed with Carnoy’s fixative (Carnoy 1887) and stained with 0.5% (v/v) orcein dissolved in 100% acetic acid.
Fluorescence in situ hybridization (FISH) Fig. 2. Time-line of the performed experiments. Time and duration of each step of the experiments is shown, together with the techniques used for checking their effectiveness. (a) Re-infection by phagocytosis; (b) re-infection by microinjection. Pg: re-suspension of recipient ciliates in homogenate of donor ciliate or in free-living P. asymbioticus culture for phagocytosis; d: days.
For FISH experiments, ciliates were fixed with paraformaldehyde 4% (v/v). FISH was then performed as described by Manz et al. (1992). Probe PnecC-16S-445 (Hahn et al. 2005), specific for Polynucleobacter belonging to subcluster PnecC (Hahn 2003) and probe Proti 445 (Vannini et al. 2012), specific for “Candidatus P. heckmanni”, were used in order to discriminate between Polynucleobacter and “Candidatus Protistobacter” (Fig. 3).
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Fig. 3. The two studied symbiotic systems. Ciliate macronucleus is visible by DAPI staining in strain FSP1.4 of E. harpa (a) and in strain strain POH1 of E. woodruffi (b). Betaproteobacterial obligate symbionts are visible by Fluorescent In Situ Hybridization (FISH) with specific oligonucleotide probes: P. necessarius inside E. harpa (c and e) and “Ca. P. heckmanni” inside E. woodruffi (d and f). Bar: 10 m.
DNA extraction and 16S rRNA gene characterization As it is not possible to discriminate unambiguously between free-living and symbiotic Polynucleobacter by FISH, 16S rRNA gene characterization was performed to this purpose. Total genomic DNA was extracted by ciliates at the end of the re-infection experiment using the NucleoSpin Plant ® II (Macherey-Nagel ) following manufacturer instructions. Amplification was then performed with the annealing taking place at 50 ◦ C × 35 cycles or by a touchdown PCR (Don et al. 1991) with the annealing taking place at 63 ◦ C, 57 ◦ C and 50 ◦ C. As the number of ciliate cells remaining at the end of the experiments was frequently very low (a few tens or even less), the amount of starting DNA represented in some cases a limiting factor. In such cases, also semi-nested PCR were performed, in order to obtain a suitable amount of amplified
DNA for sequencing. PCR products were directly sequenced in order to obtain at least the first part of the 16S rRNA gene, where several single-nucleotide differences between the gene sequences of free-living and symbiotic Polynucleobacter are located. Primers used for PCR and for direct sequencing are reported elsewhere (Vannini et al. 2012). In one case, sequencing demonstrated that in one well (i.e. in one single Euplotes cell) the original symbiotic P. necessarius survived the penicillin treatment. This well was thus excluded from further analysis. The obtained sequence has been deposited in the European Nucleotide Archive database with the accession number LT718446.
Statistical analysis Ciliate cells which died within 24 h after the re-infection procedure were excluded, taking into account the possi-
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bility that some ciliates could have been damaged by the setting-up protocols (i.e. by microinjection). The ANalysis Of SIMilarity (ANOSIM) was used to evaluate the statistical significance of observed differences between groups of data using the software PAST (Hammer et al. 2001).
Results Antibiotic treatment The used antibiotic treatment was effective, as the absence of the symbiotic bacteria from the host cytoplasm at the end of the five-days period was confirmed by aceto-orcein observations. As the only exception, at the end of the counting period in the re-infection by phagocytosis experiment, where E. harpa was tentatively re-infected with the free-living Polynucleobacter, the presence of bacteria in the host cytoplasm of some ciliates was observed. The 16S rRNA gene characterization cleared that the original P. necessarius symbiont survived the antibiotic treatment. For this reason, this experiment was repeated a second time and only results from the repetition were used for further analysis and discussed.
Re-infection experiments by phagocytosis Re-infection experiments by phagocytosis with E. harpa as recipient cell. Control ciliates (symbiotic host cells) maintained their symbionts and regularly divided as expected during the counting period, reaching after 8 days an average number of cells per well of 12.5 ± 11.6. Expected results were also obtained for the aposymbiotic recipients treated with Penicillin-G, which did not divide (only one division in one well). They reached after 8 days an average number of cells per well of 0.3 ± 0.5, as some ciliates died during the last days of counting. E. harpa cells re-infected with their original P. necessarius ingested the mixed bacteria by phagocytosis: the presence of P. necessarius inside the phagocytotic vacuoles was demonstrated by FISH with the probe PnecC-16S-445 (Fig. 4) at 48 h after mixing with homogenate. Nevertheless, no P. necessarius were present neither in the vacuoles nor in the cytoplasm later, at the end of the growth check. At the end of the growth check, ciliates of this experimental group reached an average number of cells per well of 1.3 ± 0.9. E. harpa mixed with “Candidatus P. heckmanni” reached an average number of ciliate cells of 3.2 ± 2.1. In this case, no positive signal of the probe specific for the given symbiont was observed neither in the digestive vacuoles nor in the cytoplasm, both after the 48 h period in homogenate and after the 8 days of growth check. Similar results were obtained when E. harpa was mixed with the free-living Polynucleobacter asymbioticus. The average number of the recipients per well after 8 days was of 0.3 ± 0.5. The Anosim test gave a highly significant result (p < 0.01 and R = 0.39). The growth rate of
Fig. 4. Re-infection by phagocytosis. P. necessarius, labeled with the probe PnecC-16S-445, inside the phagocytotic vacuoles of reinfected E. harpa cells at 48 h after mixing with homogenate. Bar: 10 m.
ciliates throughout the entire counting period are reported in Fig. 5a. Re-infection experiments by phagocytosis with E. woodruffi as recipient cell. Control E. woodruffi ciliates (symbiotic host cells) maintained a high division rate, reaching an average number of cells of 38.7 ± 9.6 at day 8 of the counting period. On the contrary, average number of cells for aposymbiotic ciliates (treated cells not mixed with bacteria) was 0.3 ± 0.5. Re-infection attempt with the original symbiont (“Candidatus P. heckmanni”) by phagocytosis did not lead to a recovery of division rate, as the average number of ciliates per well at the end of the counting period was of 1.8 ± 1.4. Similar results were obtained for re-infection attempts performed with symbiotic and free-living Polynucleobacter (2.3 ± 2.2 and 2.1 ± 1.3 average cells per well, respectively). The presence of bacteria inside the ciliates was never detected by FISH, both after the 48 h period in homogenate and after the 8 days of growth check. The Anosim test gave a highly significant result (p < 0.01 and R = 0.60). The growth rate of recipient E. woodruffi are reported in Fig. 5c.
Re-infection experiments by microinjection Re-infection experiments by microinjection with E. harpa as recipient cell. Control and aposymbiotic E. harpa cil-
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Fig. 5. Growth rates of recipient ciliates. Number of ciliates counted during the eight-days counting period after the re-infection attempts. C: control ciliates; AC: aposymbiotic (penicillin-treated) ciliates; Pn: ciliates re-infected with P. necessarius; Cph: ciliates re-infected with “Ca. P. heckmanni”; Pa: ciliates re-infected with P. asymbioticus. (a and b) E. harpa as recipient ciliate; (c and d) E. woodruffi as recipient ciliate. (a and c) re-infection by phagoytosis; (b and d) re-infection by microinjection.
iates were represented by the same experimental groups tested for the previous experiments (see above). Recovery of division capacity was observed if E. harpa were injected with their original P. necessaries endosymbionts, as the average number of cells per well at the end of the 8 days counting period was of 29.1 ± 8.3. In this case, the presence of P. necessarius symbiont inside the cytoplasm was confirmed by the FISH experiments. On the contrary, re-infection attempts with “Candidatus P. heckmanni” and with the free-living Polynucleobacter strain did not result in a recovery of the fission ability (average number of ciliates per well at the end of the counting period was of 0.5 ± 0.5 and 0.6 ± 1.1, respectively). In these two experimental groups, the presence of bacteria inside the ciliates was never detected by FISH. The Anosim test gave a highly significant result (p < 0.01 and R = 0.50). The growth rates of the recipients are reported in Fig. 5b. Re-infection experiments by microinjection with E. woodruffi as recipient cell. Control and aposymbiotic E. woodruffi ciliates were represented by the same experimental groups tested for the re-infection by phagocytosis
experiments (see above). E. woodruffi cells injected with their original symbiont (“Candidatus P. heckmanni”) reached after the 8 days of the counting period an average number of ciliates per well of 0.9 ± 1.4. At the end of the experiment, the symbiotic bacteria were not observed inside the cytoplasm of the recipients by FISH. When re-infection by microinjection was performed with symbiotic P. necessarius, an average number of cells per well of 2.1 ± 1.8 was obtained and no bacteria were detected inside the re-infected ciliates at the end of the experiment. The same datum for re-infection with the free-living P. asymbioticus strain was of 0.6 ± 0.9 and FISH detection gave a negative result as well. The Anosim test gave a highly significant result (p < 0.01 and R = 0.63). The number of ciliates counted in the glass wells are shown in Fig. 5d.
Discussion Success of re-infection experiments was verified by checking the presence of the used bacterium inside the recipient
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ciliates and by monitoring the growth rate of the recipients. We chose to monitor the growth rate of the recipient cells, instead of just checking their ability to perform any division (even just one more), for two reasons: (1) when Euplotes hosting “Candidatus P. heckmanni” are deprived of their symbionts, they do not always lose immediately and completely the capacity of dividing (Vannini et al. 2012), (2) it has been shown that even Euplotes naturally hosting Polynucleobacter symbionts can be able to perform one or two divisions after being deprived of the symbionts (Vannini et al. 2007). Our results confirm anyway that the growth capacity of the Euplotes species harboring Polynucleobacter or “Candidatus P. heckmanni” is indissolubly linked to the presence of the symbionts. Indeed, whenever bacterial symbionts were absent, we never detected a growth rate resembling that of the control groups. Ciliates without bacteria are definitively not able to grow. Although all the experimental groups showed a high variability, this datum is clearly evident. In some of the experimental groups where a growth recovery was not achieved, a certain degree of mortality was also observed, still confirming previous results about the obligate nature of these symbioses. As the rapidity of the effect of the antibiotic treatment can be highly variable, a five-days Penicillin-G treatment was necessary in order to obtain an efficient removal of symbionts. The lack of success of at least some of the re-infection experiments could have been also influenced by the timing. Indeed, re-infection experiments were always performed immediately after the end of the treatment, but at present it is not possible to exclude that a critical time exists after which reinfection could be no longer possible. The development in the future of a quicker method for symbiont removal, not affecting the host health, could maybe allow to verify this hypothesis. Results of the attempts of re-infection by phagocytosis showed that, in the used conditions, none of the bacteria is able to successfully colonize the host in this way. Such result is different from the ones previously obtained by Heckmann in 1980, who successfully obtained re-colonization by phagocytosis after antibiotic treatment. In that case, due to the former unavailability of adequate techniques, no check at the end of the experiments was performed in order to verify the true identity of the bacteria inside the ciliates. On the basis of our experience, we believe that, possibly, in that case, the original symbionts were not always completely removed by penicillin treatment. As an alternative explanation, it should be taken into account that those previous experiments were performed using different combinations of bacterial and ciliate strains and this aspect could indeed affect the obtained results (see also below). In our tests, ingestion of the given bacteria was demonstrated at least in one case by FISH experiments, but no re-colonization of the host cytoplasm occurred, even when the ciliates were exposed to the their original symbionts. As it is hard to imagine a different way of entrance for the establishment of the association under natural condi-
tions, we can hypothesize that colonization by phagocytosis can succeed only if specific requirements are satisfied under contingent circumstances. First of all, only a few free-living Polynucleobacter (PnecC) species or genotypes could display the physiological traits required for switching to the symbiotic life-style. Indeed, it has been recently shown that ecophysiological and genomic traits of free-living Polynucleobacter strains can present conspicuous differences (Hahn et al. 2016a). Such variability could considerably lower the percentage of free-living Polynucleobacter which, at a certain time and in a certain place, can be able to colonize an Euplotes host entering by phagocytosis. For example, non-functional secretion system genes of horizontal origin have been found in the genome of a symbiotic Polynucleobacter (Boscaro et al. 2013), but they are not present in the genome of the free-living Polynucleobacter strain QLW-P1DMWA-1T (Hahn et al. 2012). Bacterial secretion system genes can be involved in the invasion process during the establishment of a symbiosis and are often found in a degraded form in symbiotic genomes (Dale and Moran 2006). These genes could have been acquired by horizontal transfer by a free-living Polynucleobacter strain and could have enabled it to colonize a ciliate host, then degenerating up to a loss of functionality. On the other side, it has been previously shown that co-ingestion with infectious bacteria can enhance the ability of free-living bacteria to colonize ciliated hosts (Fokin et al. 2004). Co-occurrence of Polynucleobacter free-living strains with highly infectious bacteria could also represent a mechanism mediating host invasion. Therefore, more than one contingent condition could actually determine the ability of a certain free-living Polynucleobacter strain in entering an Euplotes host and switching to the symbiotic life-style. Such hypotheses could explain from one side the observed present inability of the symbionts to re-colonize by phagocytosis even their original host, and, on the other side, the unsuccessful attempts of colonization by the free-living Polynucleobacter strain QLW-P1DMWA1T . In the natural environment, free-living Polynucleobacter (PnecC) bacteria are ubiquitous in freshwater systems with large populations and many different genotypes or species coexist in the same habitat (Jezberova et al. 2010; Jezbera et al. 2011). A high frequency of phagocytosis by freshwater ciliates is therefore absolutely plausible. This could greatly improve the probability for an Euplotes host to match sometimes a compatible Polynucleobacter strain under the right conditions for the establishment of a symbiotic association. In other words, while the wide range of possible matches between potential hosts and bacteria in the natural environment can occasionally lead to the successful establishment of new symbioses, it could be much harder to obtain the same result under experimental, standardized conditions. Re-infection attempts by microinjection allow to skip the first stages of invasion of the host cytoplasm by introducing the bacteria directly inside the host cell. Nevertheless, this kind of re-infection succeeded only in the case of the
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symbiotic Polynucleobacter re-introduced inside its original host (E. harpa). The microinjection technique did not allow a re-colonization of ciliates neither when cross-infection experiments were performed (i.e. infection of a different host by symbiotic bacteria: symbiotic Polynucleobacter injected in E. woodruffi and “Candidatus P. heckmanni” injected in E. harpa), nor when free-living Polynucleobacter were used. Such results point to a high degree of specificity in the interactions between each symbiont and its host even after the invasion step, largely limiting the possibility of obtaining artificially new host/bacteria symbiotic combinations. A coevolutive pathway of reciprocal adaptation in each specific symbiotic association could indeed explain the matter. On the other side, the failure of re-infection by microinjection of the symbiont “Candidatus P. heckmanni” inside its original host (E. woodruffi) is actually harder to explain. The easiest explanation could maybe reside in a higher degree of sensibility of this bacterium toward the microinjection technique. On the other side, the high complexity of the evolutive history of this association could maybe lead to a different hypothesis. The hypothesis that “Candidatus P. heckmanni” could represent the bacterium that first established the association with Euplotes has been proposed (Vannini et al. 2012). In addition, it has been demonstrated that the symbiosis with Polynucleobacter bacteria has been established repeatedly by different, independent evolutive events (Vannini et al. 2012). This means that the evolutionary path of “Candidatus P. heckmanni” as endosymbiont in Euplotes could have been longer with respect to that of Polynucleobacter symbionts. Moreover, different Polynucleobacter symbionts became endocytobionts at different times, displaying nowadays different degrees of coevolution with their specific host ciliate. Therefore, their re-colonization abilities could be widely diverse. This could also explain previous results obtain by Fujishima and Heckmann (1984) while performing trans-infection experiments by microinjection. They used several different strains and species of Euplotes with their obligate symbionts. Indeed, in that case, the cross-infection attempts succeeded only in a few cases, but not all. The more ancient evolutionary history of “Candidatus P. heckmanni” as symbiont could justify its complete inability of re-establish a stable association, even within its original host. Similarly, among the variety of Polynucleobacter symbiotic strains, different degree of re-colonization ability could have been conserved, thus explaining why we found the Polynucleobacter symbiont from E. harpa strain FSP1.4 able to re-colonize only its original host and only by microinjection procedure. Indeed, different degree of infection ability are shown even by highly infective symbiont of ciliates, like, for example, Holospora spp., when different combinations of host’s and symbiont’s strains or species are used (Fokin et al. 2005; Fujishima 2009; Fujishima and Fuijita 1985; Nidelet and Kaltz 2007). A wider set of data on symbiotic and freeliving bacterial strains, both from genomic and physiological approaches, could help to solve the matter in the future.
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Acknowledgements This work was supported by the European Commission FP7-PEOPLE-2009-IRSES project CINAR PATHOBACTER (247658, mobility support to CS), the University of Pisa (PRA 2016 58), and by the Japan Ministry of Education, Culture, Sports, Science and Technology, TOKUBETSUKEIHI, to MF. Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. S. Gabrielli and V. Boscaro are gratefully acknowledged, respectively, for help with graphic artwork and useful critical discussion of results.
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