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Protistology 8 (1), 8—15 (2013)
Protistology
Heat shock proteins of free-living ciliates and their impact on cell adaptation to salinity stress
Alexei Smurov1, Yulia Podlipaeva2, Sergei Skarlato2 and Andrew Goodkov2
1 Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia
2 Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia
Summary
The paper describes the comparative analysis of our own experimental data concerning the chaperone activity of 70 kDa heat shock protein (HSP70) in three free-living ciliate species, Paramecium jenningsi, Tetrahymena pyriformis, Paramecium nephridiatum, belonging to the eufreshwater, metafreshwater and euryhaline ecological groups of unicellular organisms. These ciliates revealed different protecting strategies and adaptive potential of their chaperon defensive system regarding to the salinity stress. We infer that the constitutive level of HSP70 content in free-living ciliates reflects the environmental conditions of locations, where from these species originated. Consequently, when evaluating the ecological importance of constitutive HSP70 level in hydrobionts, it is necessary to take into account not only contemporary conditions of their existence, but the chaperone system “memory” as well.
Key words: chaperone system, free-living ciliates, heat shock proteins, HSP70, ecological groups of hydrobionts, Paramecium nephridiatum, Paramecium jenningsi, Tetrahymena pyriformis, salinity adaptation
Introduction
All living organisms respond to various environmental stresses by activation of different protecting mechanisms. At the cellular level, a basic and the most evolutionary conserved defensive strategy is the synthesis of stress proteins, or heat shock proteins (HSPs). These proteins are represented by a number of families, differing in molecular weight, nucleotide sequences of their genes, and functions. The best studied HSP family is characterized by the
molecular weight of about 70 kDa (HSP70). This family contains highly conservative proteins with a low species-specificity (Feder and Hofmann, 1999; Margulis and Guzhova, 2000, 2009).
In free-living protists, HSP70 are rather poorly studied in comparison to those of bacteria, parasitic protozoans from homoiothermal hosts and multi-cellular organisms. There are just a few studies of HSP70 in the non-parasitic species of ciliates (Fink and Zeuthen, 1980; McMullin and Hallberg, 1987; La Terza et al., 2001, 2004; Hori and Fujishima,
© 2013 The Author(s)
Protistology © 2013 Protozoological Society Affiliated with RAS
2003; Smurov et al., 2007; Podlipaeva et al., 2008; Goodkov et al., 2010), flagellates (Drzymalla et al., 1996; Barque et al., 2000), and amoebae (Kalinina et al., 1988; Podlipaeva, 2001; Podlipaeva et al., 2006; Podlipaeva and Goodkov, 2009; Goodkov et al., 2010). Moreover, in these unicellular organisms, HSP70 were investigated mainly during adaptation to the changes in the environmental temperature.
However, as HSPs do bear the protective function, their participation in the process of adaptation to various adverse environmental factors may be hypothesized. In particular, an important topic is the adaptation of protists — the primarily aquatic creatures — to the changes in the water salinity. Salinity, alongside with temperature, is a key factor in the evolution of aquatic organisms.
There are four ecological groups of protistan species depending on their salinity preferences (Smurov and Fokin, 2001; Kudryavtseva et al., 2007). The first group — stenofreshwater species — includes organisms that can survive in the medium with the salinity not higher than 2.5-3 %c. The second group — eufreshwater species — includes organisms surviving in the salinity range of 0 — 6-8.5 %. The species of the third group — metafreshwater ones — can persist in more salty water, up to 12-16 %. And finally, the species of the fourth group may be termed as euryhaline ones — they can sustain the direct transfer from marine to fresh water.
Classification given above deals with the so called potential ranges of salinity tolerance (Smurov and Fokin, 2001), which describe the entire amplitude of salinity values, where the species can exist, or which the species might be acclimated for. It is necessary to point out that the value of the range of salinity tolerance of the cells acclimated to some specific salinity value is always narrower than the potential range of salinity tolerance of the species.
Similar ecological groups can be distinguished within the certain large taxa of multicellular invertebrate hydrobionts (Aladin, 1996). As the origin of such groups results from the prolonged evolution history, one may presume that there are some specificities of the chaperone system functioning in the species from each group. Earlier we have already shown that ciliates react to thermal and salinity shocks in different ways (Plekhanov et al., 2006), and that the reaction to shock, expressed in alterations of HSP70 level in the cells, may be different in ciliates belonging to different ecological groups (Smurov et al., 2007).
The existence of different ecological groups of protists within certain salinity ranges presumes that the species belonging to these groups are preadapted to salinity changes within these ranges; nevertheless,
all of them can inhabit the same medium, i.e. the fresh water medium. If so, the physiological cell mechanisms, and the chaperone system among them, must also be preadapted to salinity changes.
The importance of salinity factor for the peculiarities of functioning of protists’ chaperone system may be detected in experiments recording the constitutive level of HSP70, its expenditure, and its induction in response to the salinity stress under constant temperature conditions in protists from various ecological groups.
The main goal of this paper was to reveal the specificity of chaperone system functioning in ciliated protists with different adaptive effectiveness regarding to salinity factor. For this purpose we use experimental data about constitutive level of HSP70 in three ciliate species, acclimated to salt-water and fresh-water medium, presented in our previous publications. When possible, the comparison with the relevant published data on multicellular invertebrate hydrobionts was carried out.
Brief description of the experiments1) and the obtained results
The ciliates used in the study were two Paramecium species, namely: P. jenningsi (strain SR1-10) and P. nephridiatum (strain SR98-1), both from the culture collection ofthe Department of Invertebrate Zoology, St. Petersburg State University (Russia), and Tetrahymena pyriformis (amicronucleate strain GL) from the culture collection of the Laboratory of Cytology of Unicellular Organisms, Institute of Cytology, Russian Academy of Sciences.
The determination of salinity tolerance range of all examined ciliates was based on the method developed earlier by A. Smurov and S. Fokin (2001). Acclimation of cell cultures to the extreme values of salinity tolerance range resulted in the shift of the range borders and in getting the cultures with new characters. New cultures were tested again, and the procedure was repeated till the potential border of salinity tolerance range was achieved and the further increase of salinity led to cells death.
The upper limit of the salinity tolerance of Paramecium jenningsi cells acclimated to freshwater conditions (0 %) was 3.25 %. For the individuals acclimated to 2 % , the upper limit of salinity tolerance was 6 %, for the cells acclimated to 3.5
1) For the detailed description of all methodical procedures see the following publications: Plekhanov et al., 2006; Smurov et al., 2007; Podlipaeva et al., 2008; Goodkov et al., 2010.
% it was 8.5 %, being thus the potential border of salinity tolerance range of this Paramecium species.
The upper limit of the salinity tolerance of Tetrahymena pyriformis was 8 % — for the cells acclimated to fresh water (0 %), 12 % — for the cells acclimated to 2%, and 15 % (potential border of salinity tolerance range) — for the cells acclimated to 10 %. Cells acclimated to 10 % salinity sustained direct transfer into fresh water (0 %), and could later adapt to it.
The upper limit of the salinity tolerance of P. nephridiatum cells acclimated to freshwater medium (0 %) was set at 20 %, and for the cells acclimated to 10 % medium — at 34 %.
Thus, the potential salinity tolerance ranges were determined: for P. jenningsi — 0-8.5 %, for T. pyriformis — 0-15 %, and for P. nephridiatum — 0-34 %. Using the classification given above, the species P. jenningsi belongs to the ecological group of eufreshwater organisms, T. pyriformis — to metafreshwater, and P. nephridiatum to euryhaline ones.
Paramecium jenningsi
In the salinity shock experiments, cells grown in the freshwater medium (0 %) were placed for
2 h in a medium with 2 % salinity. The cultures acclimated to 2 % salinity were placed in fresh water also for 2 h. Protists cultivated in the media with the initial salinity values were used as the control; the culture density was the same in the control and in the experiments (Table).
In the total protein extract of the eufreshwater ciliate P. jenningsi, an antigen cross-reacting with anti-HSP70 antibodies was revealed. It was shown by the method of dot-blotting that the level of HSP70 in P. jenningsi cells grown in the freshwater medium was very low, but it increased after salinity shock (Plekhanov et al., 2006). On the contrary, ciliates acclimated to 2 % demonstrated a high constitutive level of HSP70, which decreased after salinity shock (Table).
Paramecium nephridiatum
P. nephridiatum cells acclimated to fresh water (0 %) were placed for 1 h into water with 10 % salinity; ciliates acclimated to10 % were placed for 1 h into fresh water (0 %); then in both cases the cells were returned to the medium with the initial salinity. These cells were considered as treated by salinity shock (Table). For the control, ciliates were
placed into water with the salinity usual for them, for the same period of time. Some of the cells were subjected to an analogous impact with the following difference: after the transfer into water with a different salinity, the cells were not returned to the medium with the initial salinity after an hour but were left in the water with a different salinity until the end of the experiment (up to 24 h). Those cells were considered as subjected to “adaptation”.
A polypeptide antigen of ca. 70 kDa was revealed by western blotting in the total protein extract of the euryhaline ciliate P. nephridiatum, both in the intact (control) cells after prolonged acclimation to the freshwater medium or the salt water medium, and in the salinity-shocked cells. Besides, P. nephridiatum cells acclimated to 10 % were shown to possess an additional polypeptide with a molecular weight of about 60 kDa, which also cross-reacted with anti-HSP70 antibodies (Smurov et al., 2007). The protein with a molecular weight of about 60 kDa was present in a considerable quantity, which was higher than that of the protein of about 70 kDa, in P. nephridiatum cells acclimated to 10 %, and was almost absent in the cells acclimated to fresh water.
The level of HSP70 in ciliates acclimated to freshwater conditions was much higher than in ciliates acclimated to 10 % (Smurov et al., 2007).
In ciliates acclimated to fresh water and salt water, salinity shock elicited an asymmetric response as to the expenditure/synthesis of HSP70. The HSP70 concentration in the cells transferred from the 10 % medium into fresh water was much higher than that in the cells transferred from fresh water into salt water, the time after transfer being equal (Table).
The level of 70 kDa protein after transfer from 10 % medium to fresh water (10 ^ 0%) was higher than that in the control. After transfer from fresh to salt water (0 ^ 10%), the HSP70 staining zone was weak, the protein concentration being much less than in the control.
“Adaptation” to a new salinity (a prolonged variant of the salinity shock) in the euryhaline ciliate P. nephridiatum resulted in HSP70 induction in the experiments with both directions of salinity changes (Table).
Tetrahymena pyriformis
T. pyriformis cells were placed for 1 h into water with a different salinity: the cells acclimated to 2 % were placed in 10 %, and the cells acclimated to 10 %, into 2 %. Then the ciliates were returned to the medium with the initial salinity. For the control,
Table. Ecological characters of ciliates, their acclimation to media with different salinities, and peculiarities of their reaction to salinity shock.
1 Ecological groups and " species under study Characters of species Eufreshwater Paramecium jenningsi Metafreshwater Tetrahymena pyriformis Euryhaline Paramecium nephridiatum
Potential borders of salinity tolerance range The scheme of ciliate acclimation Salinity shock (1h, then return to initial medium) Induction (+) or expenditure (-) of HSP70 after the stress, the absence of pronounced reaction (+/-) to the stress G-8.5%o G^2%o (I) G^2%o (II) 2^G%o (I) + (II) - G-15% G^2^1G%o (I) 2^1G%o (II) 1G^G%o (I) +/- (II) +/- G-34% G^1G%o (I) G^1G%o (II) 1G^G%o (I) - (II) +
ciliates were placed for the same time into water with the salinity usual for them (Table).
In the metafreshwater ciliate T. pyriformis, two zones were revealed in the control cells cultivated under optimal conditions (0 %): an intensely stained zone with a molecular weight of about 70 kDa (“72 kDa”) and a zone of about 60 kDa (Podlipaeva et al., 2008). In the intact T. pyriformis cells acclimated to 2 %, a stained zone of a somewhat larger molecular weight (“73 kDa”) was revealed, which remained almost unchanged in 2, 4 and 24 h after the salinity shock during 1 h at 10 %. Besides, both in the control samples and in the samples prepared in 24 h after salinity shock, a weakly stained zone corresponding to a protein with a molecular weight of about 65 kDa was revealed.
T. pyriformis acclimated to 10 % had a constitutive HSP with a molecular weight of “72 kDa”, which had the same position as in the ciliates from fresh water, and was located a little bit lower than that of the ciliates acclimated to 2 %. The level of this protein decreased in 2 h after salinity shock at 2 %. In 4 h after shock, the blot contained a protein with a slightly higher molecular weight (“73 kDa”), which substituted the protein with molecular weight about 72 kDa. Finally, in 24 h after shock both zones, “72” and “73 kDa”, were distinctly stained on the blot (Podlipaeva et al., 2008). The protein with molecular weight of about 65 kDa was not found.
No induction of HSP70 synthesis in the meta-freshwater ciliate T. pyriformis cells was registered after salinity shocks in both directions of salinity changes (Table). Moreover, in the cells acclimated to 10 %, there was a noticeable expenditure of the “72 kDa” protein after salinity shock. It is worth mentioning that in T. pyriformis the differences both in spectra and levels of content of constitutive HSP were revealed depending of the salinity of their long term acclimation.
To sum up, eufreshwater and euryhaline species of the genus Paramecium have different strategies of
the chaperone system’s response to increasing and decreasing salinity. In the eufreshwater species P. jenningsi, acclimation to increased salinity results in an increase of the constitutive level of HSP70 in the cell, i.e. an acclimation to such a slight shift (from 0 to 2 %) of salinity is a stress for this species. Acclimation of the euryhaline species P. nephridia-tum to increased salinity does not result in any change in the HSP70 level. Decreasing salinity of the medium (from 2 to 0 %) is accompanied in eufreshwater P. jenningsi by the decreased, whereas in euryhaline P. nephridiatum — by the increased level of HSP70 in the cell. In P. nephridiatum individuals acclimated to fresh water (0 %), the level of HSP70 was much higher than in P. nephridiatum individuals acclimated to salt water (10 %). So, for euryhaline ciliates, a prolonged acclimation to fresh water is a stronger stress than acclimation to 10 %. Prolonged acclimation to fresh water appeared to result in the activation of their chaperone system.
Therefore, in fresh water these ciliates turn out to be pre-adapted to abrupt changes in the environmental salinity and respond to them by using the pool of HSP accumulated in the cell. It is, for example, expressed in the decrease of high HSP70 level in ciliates acclimated to fresh water (0 %) after their transfer to salt water (10 %).
As for the reaction of P. nephridiatum to salinity shock, the HSP70 concentration increased in the cells transferred from the 10 % medium into fresh water and decreased in the cells transferred from fresh water into salt (10 %) water. Thus, the chaperone system of euryhaline ciliates, such as P. nephridiatum, is characterized by an asymmetric response to salinity variations, namely, by the expenditure/synthesis of HSP70 after salinity shock, depending on the direction of salinity changes.
“Adaptation” to a new salinity (that is, a case of the prolonged salinity shock) brings about HSP70 induction in P. nephridiatum, whatever direction the salinity changes. This observation agrees with
the reports that in some instances only a prolonged stress elicits HSP70 induction. For example, in the ciliate Moneuplotes crassus the level of HSP70 was the same as in the intact cells after a short heat shock (5 min) but increased after a prolonged heat shock, reaching the maximum after 180 min (Ullmann et al., 2004).
It is worth mentioning that the data about prolonged stress in unicellular hydrobionts do not totally correspond to analogous data on multi-cellular ones, such as marine molluscs. For example, HSP70 concentration in the cells of Chamelea gallina (Bivalvia) changed only in the cases of mollusc adaptation to low salinities, and in digestive gland it was increased, but in the gills — decreased (Monari et al., 2011). It was also shown that the level of HSP70 in gill epithelium ofmussels Mytilus edulis (Bivalvia) increased after long term acclimation to both high and low salinities, but prolonged salinity stress of 24 h caused HSP70 induction only in low salinity (Podlipaeva and Berger, 2012).
As we stated above, in the metafreshwater ciliates Tetrahymena pyriformis no any induction of HSP70 synthesis was discovered in organisms after salinity shocks in both directions of salinity change. On the contrary, there was a noticeable expenditure ofthe 72 kDa protein, which substituted the 65 kDa protein 4 h after the shock. Thus, we can conclude that the chaperone system of T. pyriformis is less mobile than that of the two Paramecium species studied in response to both a prolonged salinity acclimation and a short stress.
It is worth mentioning that metafreshwater Tetrahymena ciliates are known to possess a heat shock protein, called HSP90, with a molecular weight of 82-85 kDa (Frankel et al., 2001). Its primary structure does not have a high homology with HSP70 ofvertebrates, whereas the protein found in our study has common antigen determinants with HSP70 of vertebrates — a fact that may indicate a considerable homology in primary structure.
To sum up the results on dynamics of HSP70 level in the ciliates treated by salinity stress conditions, it may be concluded that unicellular organisms belonging to eufreshwater and euryhaline (but not to metafreshwater) groups ofspecies respond to salinity shock at first by decreasing and then by increasing concentration of HSP70 in the cells.
Discussion
Studies of HSP70 content in the cells of invertebrates from natural populations show that slight alterations of environmental temperature
do not result in changes of HSP70 level (Feder and Hofmann, 1999). Long term (about a month) trends of environmental temperature fluctuations are essential for noticeable change of constitutive HSP70 level in the cells (Menge et al., 2002). Besides, it was shown that there is a positive correlation between HSP70 level in the cells of insects and the border-values of their temperature tolerance ranges (Evgen’ev et al., 2007). The similar data were obtained when studying temperature adaptations of hydrobionts. Reliable positive correlation was revealed between the environmental temperature and the level of HSP70 content in the gills of Mediterranean mussels Mytilus galloprovincialis. Moreover, pronounced and reliable seasonal changes in protein content in these mussels were observed following environmental temperature changes (Hamer et al., 2004).
Our experiments differ from the investigations mentioned above where the animals mainly were obtained directly from their natural habitats; the ciliates under study underwent the same conditions in terms of medium (all of them were preliminary acclimated) and culture growth phase. Ciliates are unicellular organisms, therefore some restrictions and reservations related to the tissue specificity of multicellular organisms are not relevant to them. All these circumstances allow evaluating impartially the ecological sense of HSP70 constitutive level in the cells in connection with their different salinity tolerance (i.e. the capability to survive in the media with different salinities).
According to our data on eufreshwater and euryhaline unicellular organisms, different strategies of the chaperone system response to increasing and decreasing salinity may be observed. Acclimation to elevated salinity results in an increase of the constitutive level of HSP70 in cells of eufreshwater ciliates, whereas in the euryhaline ones it does not result in any change in the HSP70 level. Decreasing salinity of the medium is accompanied by an increased level of HSP70 in both euryhaline and eufreshwater species of Paramecium ciliates, and the metafreshwater species Tetrahymena pyriformis which is “intermediate” according to its ecological characters, reacts weekly to salinity changes in the medium. The level of HSP70 content in T. pyriformis cells practically does not allow presuming that its chaperone system is less reactive than that of eufreshwater and euryhaline ciliate species. We cannot exclude that constitutive level of HSP70 in the cells ofthese ciliates, even belonging to different ecological groups of species depending on their salinity tolerance, may reflect, in some respect, the conditions in the habitat of their origin: fresh water
bodies in the case of P. jenningsi, fresh or brackish water bodies in the case of T. pyriformis, brackish-water estuaries or seas in the case of P. nephridiatum. Differences in the salinity tolerance limits of the species studied suggest that, even though they are pre-adapted to salinity changes within these limits, all of them may live in the same medium — fresh water.
Thus, we presume that when evaluating the ecological importance of constitutive HSP70 level in the unicellular hydrobionts it is reasonable to take into account not only the current conditions of their habitat, but also the “memory” of their chaperone system. The experiments with preliminary acclimation represent a convenient method to reveal such a “memory”.
One of the examples of chaperone system “memory” is the induction of HSP synthesis (HSP70 among them) in the trypanosomatid flagellate Phytomonas characias, a parasite of plants, after temperature shift from 22 to 37 oC (Sanchez-Moreno et al., 1997). Nevertheless, these trypanosomatids do not have warm-blooded host in their life cycle, HSP70 synthesis induction in Phytomonas cells occurs namely at 37 oC, which is a characteristic feature of heteroxenous trypanosomatids (Trypanosoma spp.), parasitizing mammals. This fact may be considered as a sort of chaperone system “memory” of flagellates from the genus Phytomonas, which is phylogenetically connected with ancestral trypanosomatids — parasites of homoiothermal hosts, and as one more confirmation of the presence of common ancestors in all representatives of this family including those turned to parasitizing insects and plants (Maslov et al., 2001).
It is known that in the multicellular organisms which inhabit highly variable environments, constitutive HSP70 level is elevated (Ulmasov et al., 1992; Evgen’ev et al., 2005; Garbuz et al., 2008). At the same time, it was shown that the deep-sea animals adapted to thermally stable conditions should be highly sensitive to temperature change and should not have inducible heat-shock responses; it concerns the cases when high constitutive levels of HSP70 are present (Berger and Young, 2006).
We obtained similar data for various protists (Podlipaeva, 2001; Plekhanov et al., 2006; Podlipaeva and Goodkov, 2009); the hypothesis was put forward that high constitutive HSP70 level may be accounted as the protists’ preadaptation not only to temperature changes, but to other factors as well (Plekhanov et al., 2006; Podlipaeva and Goodkov, 2009). The presence of similar ecological groups for uni- and multicellular organisms, distinguished
according to their relation to environmental salinity changes (Smurov and Fokin, 2001), allow presuming the universality oftheir chaperone system reactions.
We have also shown that after certain environmental salinity changes, eufreshwater and euryhaline ciliate species may demonstrate pronounced HSR (heat shock response). Species with intermediate adaptive capacities (e.g. T. pyriformis), on the contrary, possess initially high HSP70 level and lack HSR. It may be hypothesized that high level of HSP70 content and the absence of HSR allow the species to exist in the extremely changeable conditions. However, in order to be able to colonize new habitats (e.g. salt water for freshwater species and vice versa) the presence of HSR is essential.
Aknowledgements
This study was funded by the Russian Foundation for Basic Research (grant 13-04-00703) and the German Federal Ministry of Education and Research 01DJ12107.
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Address for correspondence: Andrew Goodkov. Institute of Cytology, Russian Academy of Sciences, Tik-horetsky Ave. 4, St. Petersburg, 194064, Russia; e-mail: pelgood1@gmail.com
