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Protistology 1 (3), 124-132 (2000) Pl'0tiSt0l0gy
February, 2000
On the methods for the estimation of salinity tolerance of ciliates
Alexei O. Smurov
Zoological Institute Russian Academy of Sciences, St.Petersburg, Russia
Summary
Critical review of research methods for tolerance and potential tolerance of ciliates is given. Using several Paramecium species as an example the original approaches to potential tolerance estimation is presented. Connection between resistance, tolerance and potential tolerance is shown. Model for explanation of the connection is proposed. Several application aspects of diagrams of the connection between acclimation salinity and value of upper salinity tolerant limit in the ciliates are considered.
Key words: acclimation, tolerance, potential tolerance, resistance, salinity,
Paramecium
Introduction
Two significant interacting ways of investigating the salinity tolerance exist. Firstly, obtained data can indicate the taxonomic relationship or distinction between taxa of low range. Secondly, evaluations of organism salinity tolerance are enabled to construct classification of organism relation to salinity impact. It is obvious that the classification will show one of principal tendencies in evolution of some protists and metazoan phyla. It is easy to see that both reasons are connected with the problem of organism evolution on the different taxonomic levels.
The logic of research of organism salinity adaptations development has rised the questions a long time ago. However, relatively complete data were obtained only for some large taxa of metazoans. Investigations resulted in the creation of classification scheme which was supported by many researchers. According to this classification scheme freshwater, brackishwater, marine, euryhaline and hyperhaline species exist. Criteria for discrimination of the categories were elaborated for freshwater and marine species of metazoans only and correspondently for faunas. Distinctions among other categories are unclear and debatable for some taxa. This is caused by the absence of any experimental investigations of species salinity resistance and tolerance. If the experimental data are absent, investigators use the presence in different salinities. Incorrectness of such point of view was clearly shown (Filippov, 1995; Orlova et al., 1998).
To solve the general classification problem one can construct convenient classification systems for the most significant taxa. At present, data on salinity tolerance for most phyla of protists are absent. However, Ciliophora is one of the most investigated protist taxa in this respect.
All data on ciliate salinity reactions mainly concerned organismic and suborganismic levels of adaptations. Most of studies on the ciliate salinity adaptations were made in the first half of the 20’s century. Methodical level of these investigations was primitive from the modern point of view. Recent publications were dedicated mainly to the mechanisms of cell osmotic adaptation and cannot be used as the basis of classification problem solution. A lot of evaluations of salinity tolerance concerning some ciliate species are based upon the data obtained in water of different salinity, which is similar to the situation with metazoan organisms. It is interesting, that most salinity investigations of the ciliate species were made on paramecia. If to analyze and to complete data on the results obtained it can be basis for solution of the problem of classification of organism relation to salinity impact and problems of reaction relationship to salinity impact for low range taxa. The first step on this way should be revision of present and elaboration of new methods for estimation of resistance and tolerance in Paramecium.
Methods of presentation and analysis of results on resistance
At present there are two methods of the lethal salinity impact evaluation. The first one is the evaluation of damage degree during standard time. The second is evaluation of latent periods of damage symptoms in different salinities. The first method is prevailing. Routine mathematical way of obtained data analysis is estimation of median (LT50), threshold (LT0) and absolute lethal (LT100) values. There are methods of their calculation in many manuals and statistic computer programs. Obtained values of LT50
© 2000 by Russia, Protistology.
or LT100 are used for construction of diagrams of connection between response time and salinity.
The hyperbolic dependence for LT50 in test toxic substance is a characteristic feature of metazoan organisms. The first extreme corresponds to 0.5-4 h of experiment, the second one to some hours for fast acting toxicants up to 8-12 days for slow acting toxicants (Lesnikov, 1979). Powers (1917) was the first to show the existence of the first extreme which was named Powers boundary. Typical equation of dependence curve of the median lethal time on the toxic substance concentration, according to Warren and Doudoroff (1971), has form:
Const = (c - a)h * (T - b), where c - concentration of toxic substance, T -median lethal time, h - constant power index for equation, a -incipient level or threshold concentration, b - threshold time little depending on toxicant concentration and characterizing “agony of organism”.
Peculiarity of lethal experiments with protists is that cells can have one or few propagation cycles at lethal concentrations of toxic substance about tolerant limit. As quantity of organisms in lethal experiment may be not constant, conventional method of tolerant limit calculation for protists is not applied. Therefore, tolerant limit can be estimated from data obtained for the concentrations, where cells can be alive 24 h or less. In this case there is only the first extreme on a curve (Powers boundary).
Function of the median lethal time dependence in test salinity have typical hyperbolic form for paramecia (Smurov and Fokin, 1999; Smurov et al., 1999). As the hyperbola has only one extreme, one can use hyperbolic line-fractional regression for its mathematical analysis.
General formula of hyperbolic linear-fractional dependence is resulted as:
aSt + b
Ltx =—‘----------
—St + d
where St - test salinity, a,b,c and d - coefficients. This formula can be presented in the other form:
b da
Ltx =- + ^-—
c
Ltx = T min +
St +------
b da a
where A = — — — - oblongness along Ltx axis, Tmin = —,
value of asymptote parallel to the test salinity axis, d
S min = —, value of asymptote vertical to the test salinity
c
axis.
It is obvious that T is equivalent to threshold time,
min A 7
S - incipient level or threshold concentration. As cells
min
do not die at threshold concentration it is possible to consider it as tolerant limit. After all changes the formula has the following more “biological” form:
Difference between Warren and Doudoroff’s formula and hyperbolic linear-fractional formula is in the exponent of h. With the help of exponent h one can describe more general curve group class than linear-fractional formula.
A different way to express the form of dependence on substance concentration is the time of mortality (50% or 100%) with representing the curve on semi-logarithmic or logarithmic diagram.
The large experimental material, obtained by researches, shows the existence of three models of this dependence (Fig. 1). The curve of mortality consists of two segments, which form an angle. In the curve of the type 1 the upper segment is displaced from the lower segment to the right and upwards. In the curve of the type 2 displacement of the segments is not observed. In the curve of the type 3 the upper segment is displaced from the lower segment to the left and down.
Displacement of the upper segment to the right reflects the existence of adaptive processes, which appear in organism under the influence of relatively delicate damaging substances. The adaptive model is the most frequent. The curve of the type 2 can be obtained after the influence of basic metabolism inhibitors depressing the adaptive mechanisms. The curve of the type 3 was obtained in the experiments with influence of high temperature on isolated animal cells and tissues. This type is uncommon. In normal system of coordinates curves of the types 1 and 3 are hyperbolae. The curve of the type 2 is exponent.
This approach was created for comparison of lower segments of animals mortality curve (Zhirmunskyi, 1959, 1969). Earlier it was believed that primary resistance, which was shown by the lower segment, had species-specific form. Mainly this method was used for investigation of thermoresistance of marine animals population. At the same time researchers of thermoresistance did not pay any attention on the position of the upper segment.
Material and Methods
The resistance and tolerance of ciliates acclimated to the media of various salinities created with marine salt were investigated. The following species were used in the experiments: P caudatum, P. putrinum, P. sexaurelia, P. bursaria, P. calkinsi and P nephridiatum (Table 1).
Cells were cultivated on lettuce medium inoculated with Enterobacter aerogenes (Sonneborn, 1970). The necessary salinity was obtained using artificial marine water (Chubravyi, 1983), diluted with lettuce medium. Before the experiment, the ciliates were maintained in freshwater or marine lettuce medium for not less than two months. The ciliate cultures were fed twice a week. The scheme of
Fig. 1. Types of narcotization (mortality) curve according to Seravin (1965). I - curve for organisms with adaptation to toxicant; II - curve for organisms without adaptation; III - curve for organisms subjected temperature impact.
Table 1. Paramecium species which were used in experiments.
Species Stock Place of the stock origin Date
P. caudatum PK-100 Russia, Stary Petershof 1995
P.sexaurelia Psex3 Stuttgart, Germany 1995
P. calkinsi OCE4-1 Russia, Keretsky Archipelago of the White Sea 1994
P. nephridiatum I1-5-4 Jerusalem Zoo, Israel 1996
P. bursaria PK-60 Russia, Stary Peterschof 1994
P. putrinum PG-5 Russia, Leningrad region, Gatchina 1995
lethal experiments was similar to that, used earlier (Smurov and Fokin, 1999).
In our investigation two approaches of tolerant limits evaluation were applied. In the first one the asymptote which is vertical to axis of a test salinity (Smin) was considered to be the tolerant limit. In the diagrams of experimental dependence time of 50 % mortality - test salinity the vertical asymptote corresponds to salinity under which cells do not die. It was obtained by calculation from the linear-fractional hyperbolic regression. The original computer program with four algorithms of linear-fractional hyperbolic regression was used.
The second approach is based on a new technique (Smurov and Fokin, 1998) which takes into account certain features of protists biology. For most unicellular organisms one could say “dividing therefore existing”, analogous to Latin sentence “thinking therefore existing”. Therefore we propose to regard as tolerant limit a value of lethal salinity not for individual cell but for cells population. In this case we take into account that individual cells subjected to salinity impact can survive and exist during
long time but cannot produce viable posterity. For experimental studies these conditions can be formulated as capability of the stock preservation. This method was used to investigate all species.
As we were interested not only in potential tolerance but also in variability of upper tolerant limit depending on salinity of acclimation, we had modified the method of stepwise acclimation. Our technique did not always use acclimation to extreme values of a tolerant range as it was offered by the authors of stepwise acclimation technique. We acclimated cells to several salinities within tolerant range. After finishing acclimation (about 2 months) tolerant limits were estimated for each salinity of acclimation. Later the procedure was repeated again for all tolerant ranges obtained. Obtained data were used to construct the diagrams of the dependence of the upper tolerant limit on the salinity of acclimation. Potential tolerance obtained by stepwise acclimation is identical to that obtained by our approach. The new method in comparison with stepwise acclimation allowed to receive the new information, which can be used for the taxonomic analysis and
Salinity, %%
Fig. 2. The curve of mortality of some Paramecium species against salinity impact.
analysis of adaptation mechanisms. Analysis of upper tolerant limit variability was made with the help of the program “STATISTICA” for “WINDOWS”.
Results
Semi-logarithmic curves of 50% mortality time in test salinity were obtained for some Paramecium species which were acclimated to freshwater lettuce medium. In our experiments on ciliates resistance to salinity impact we have managed to obtain curves of the types 1 and 2 for all investigated paramecia species (Fig. 2). P sexaurelia, P bursaria and P putrinum have resistance of the type 2. The other paramecia have resistance of the type 1.
P putrinum cannot be adapted to salinity above 2.4%o. As it is shown at the diagram (Fig. 3) this species cannot be acclimated at all. In all experiments the upper salinity tolerance limit remained constant for every salinity.
Another result was obtained for P bursaria. With increase of acclimation salinity the resistance of cells grew linearly. However, subsequent long time cultivation in new salinities was not possible in all cases. Diagram of upper limit obtained as the result of cultivation during 2 months is displaced lower than diagram corresponding to 7 days of experiment (Fig. 3). In fact, before death individual cells could exist for about a month in 3.5-4% and have several cycles of division. For the second day of experiment all the cells died in salinities above 4.5% without dependence on the salinity from which they were transferred.
The upper limit of tolerance of P. caudatum grows linearly up to 3% salinity of acclimation. The maximal salinity to which it was possible to adapt this stock was 7% (Fig. 4). P sexaurelia can live in the wider limits than
P caudatum. The maximal salinity in which stock can live during unlimited time is 15%. This species can survive in greater salinity during 7-9 days. In this case cell division usually does not occur.
Data on vertical asymptotes and tolerant limits obtained using stock preservation method of P nephridiatum acclimated to salinities 3%, 5%, 10% and fresh water are presented on Fig. 5. As the diagram shows, all asymptotes have different values. Comparison with data obtained using other method for tolerant limit evaluation has shown that both methods are almost identical.
The value of extreme of mortality curve of upper salinity tolerant boundary and potential salinity limit for some Paramecium species are summarized in Table 2.
Discussion
The terms of tolerance range and potential tolerance range, which are widely used in the studies of salinity tolerance of the metazoan organisms have different meanings. The tolerance range is the range of salinity between the upper and lower lethal limits. It was supposed that certain physiological functions of an organism can be disturbed but it does not die within tolerance range after immediate changes. Disturbed functions of an organism are recovered partially or fully during the process of acclimation (Filippov, 1998).
The term of potential tolerant range is connected with an essence of acclimation idea, which assumes that the tolerance range depends on previous salinity conditions (existence of experimental matter conditions). The tolerant range and the values of limits can be reversible in the acclimation process. As the result of consistent acclima-
Salinity of acclimation, %o
Fig. 3. Upper salinity tolerant limits. 1 - upper tolerant limit of P. bursaria after 7 days of acclimation; 2 - upper tolerant limit of P. bursaria after 60 days of acclimation; 3 - upper tolerant limit of P. putrinum after 60 days of acclimation.
Salinity of acclimation, %o
Fig. 4. Upper salinity tolerant limits. 1 - upper tolerant limit of P. sexaurelia after 7 days of acclimation; 2 - upper tolerant limit of P. sexaurelia after 6O days of acclimation; З - upper tolerant limit of P. caudatum after 6O days of acclimation.
tion to increasing factor the organism will be capable to survive in more wide range than initially. Continuing acclimation in the direction of reduction or increasing one can obtain the value of salinity when subsequent acclimation is impossible.
Estimation of salinity tolerance of various metazoan and protist species and its comparison is difficult because at present there are no conventional methods of its deter-
mination. For last 30 years many papers devoted to the estimation of potential salinity tolerance of metazoan organisms were published. According to Filippov (1998), who made a critical review of these investigations, there are 4 principal methods of evaluating potential tolerance: direct transfer, methods of physiological adaptation, stepwise acclimation and express method based on the analysis of salinity tolerance of isolated tissues.
Salinity of acclimation, %
Fig. 5. Upper salinity tolerant limits of P. nephridiatum. 1 - data obtained by the resistance method; 2 - data obtained by the stock preservation method.
Table 2. The value of extreme of mortality curve of upper salinity tolerant boundary and potential salinity limit for some Paramecium species.
Species Tolerant limit, %o Breakpoint, %o
P. caudatum freshwater - 7 З
P. sexaurelia freshwater - 15 5.5
P. nephridiatum freshwater - 40 4.5
P. bursaria freshwater - 3 0.5
P. putrinum freshwater - 2.4 -
When the method of direct transfer is applied, salinity in experimental aquaria (microaquaria) with the test organisms is changed sharply. The state of organisms in the conditions of different salinity is estimated by the percentage of mortality in unit time, functional activity and other parameters. Approach to analysis of the obtained data and the final result do not differ from analogical methods of tolerance estimation. Correspondently, this method does not work for evaluation of potential tolerance.
The method of physiological adaptation is based on the results of prolonged observation of test organisms. It is shown that many physiological processes of organism normalize during 2 days in small salinity modifications. It is offered to change salinity for 2% every two days (Karpevich, 1947). Thus, the results obtained by this method showed that salinity range was significantly wider than in experiments with direct transfer (Karpevich, 1953,
1958; Bekmursaev, 1970, 1971). Assumption that about 2 days are necessary to accomplish the acclimation is an essential disadvantage of the method. In the later investigations it is shown that as a rule more time is necessary to finish the acclimation (Khlebovich and Kondratenkov, 1971; Filippov, 1995; etc.).
The most modern method of potential salinity tolerance evaluation is the stepwise acclimation developed in 70’s (Khlebovich and Kondratenkov, 1971, 1973). During stepwise acclimation minimal duration of acclimation to next salinity is 2 weeks. For determination of salinity step for each stage of experiment the tolerant range of previous stage of experiment is taken into account. As it was shown by investigations the stepwise acclimation is most preferred for studing of potential salinity tolerance of meta-zoan organisms (Filippov, 1995). Basis of the method is a large actual experimental material obtained on metazoan
and protists organisms. Acclimation to different factors of environment is finished about 10-14 days. The duration of the total experiment is essential disadvantage of this method. Sometimes experiment continues up to 4-6 months.
The express method based on the analysis of salinity tolerance of isolated tissues is used for metazoan animals only. As protists have no tissues this method is not useful for protists. Hence, this method is not discussed below.
Most part of investigators tested ability of paramecia to adapt themselves to marine water by the direct transition (Balbiani, 1898; Zuelzer, 1910; Heres, 1922; Adolph, 1925; Chatton and Tellier, 1927; Yokom, 1934). Brackishwater P. calkinsi and P. woodruffi and freshwater P caudatum and P aurelia were used in their experiments. Freshwater species endured direct transition into the salinity up to 3-7.5 g/l. P calkinsi can endure transfer into salinity of 30-31 g/l. P woodruff endured direct transition into 10 g/l only (Bullington, 1930).
Some investigators mentioned above in their experiments used the method, which can be classifed as physiological adaptation (Balbiani, 1898; Zuelzer, 1910; Chatton and Tellier, 1927). According to their data P. caudatum managed to adapt itself to 10 g/l.
Finlay (1930) in his experiments on P caudatum, P. aurelia and Frish (1935, 1937, 1939) for P caudatum, P. multimicronucleatum actively used the method of gradual salinity increase. The gradual increase of salinity in their experiments was obtained by free evaporation of cultural medium. The method elaborated by Finlay and used by Frish had distinct disadvantage. It was caused by the necessity to correlate evaporation of culture medium and feeding of ciliates during long term experiment.
The method of stepwise acclimation which was considered to be the best in early studies of potential salinity tolerance of ciliates was never used.
Since values of salinity tolerance boundaries can be determined for species having mortality of the second type with the other method we suppose that curve of mortality must have an extension. It means that upper segment of curve must be situated within the narrow field of test salinity values. This part of curve is almost perpendicular to the test salinity axis. To obtain this result it is necessary to carry out experiments with very fractional values of test salinities. The upper part of the curve is not revealed. It is connected with technical errors of experimental methods. As a result it is possible to conclude that the curve of the type 2 in fact is related to the type 1 i. e. with respect to salinity there is only one type of mortality necessarily connected with adaptation.
This result confirms Seravin’s conclusion (1965) that the curves of the types 2 and 3 are derived from the curve of the type 1. It is possible to assume that curves of the type 1 were considered by many researches “wrongly” as the curve of the type 2. It is interesting, that development
of adaptation processes reflecting the upper segment of the curve of the type 1, resulted in achievement of tolerant limit. It can be indirect confirmation that mechanisms of adaptation increasing resistance of cells are acclimational. If it is correct, the beginning of the action of acclimation mechanisms can be easily determined. The point of excess between the upper and lower segments of the curve of the type 1 corresponds to this moment.
It is necessary to notice interesting difference of pro-tist salinity reactions from those of metazoan animals: they can reproduce at the same time in salinities outside tolerant limit. Metazoan animals behave vice versa as a rule. According to the examples given by Kinne (1957), they stop to reproduce long before the tolerant limit is achieved.
According to the interpretation of Warren and Doudoroff model for P. putrinum and P. bursaria, it is easy to interpret diagrams of upper tolerant limit. Acclimation to increasing salinity is impossible for P putrinum, therefore existence in weakly saline medium can result in increasing of “standard agony time” (threshold time). In this case change of tolerant limit and prolongation of curve do not happen. This increase of “agony time” was noticed for P bursaria also. These cells do not die after cultivation during one week in new salinity, which could be explained only by the fact that the curve of mortality for this species acclimated to different salinities must have two extremes.
The important feature of the left part of the diagrams (Figs 3, 4) is the change of tolerant limit which has approximately linear form. In the modern literature on salinity adaptations this phenomenon is considered as the proof that experimental animals really have been acclimated (Khlebovich, 1981).
As a result of increasing salinity of acclimation the tolerant limit changes to the certain ranges. For all species there is a value of acclimation salinity when value of tolerant limit does not change. The right part of diagram is parallel or about parallel to the abscissa axis (Figs 3-5). The function, according to this diagram, is described by breakpoint regression successfully. Therefore variable Smin from Warren and Doudoroff’s formula can be replaced with
(m+m* S ) * (S . <n)+(m+m* S ) * (S . > n),
v 01 11 a7 v min 7 v 02 12 a7 v min 7’
where S - tolerant limit value, S - salinity of accli-
min a
mation, n - extreme, m01, m11, m02, m12 - coefficients of equation. Role of extreme point is not mathematical only. There is biological meaning of its existence and value.
Now there are a few investigations concerning analysis of external and internal osmolarity in ciliates. The data obtained by Stoner and Duncham (1970) for Tetrahymena pyriformis specify ability of this species to regulate the internal osmolarity in the limits up to 5 %. Apparently, all ciliates species of freshwater origin have this ability.
Internal medium is hyperosmotic in relation to external not only in freshwater Tetrahymena pyriformis, but also in marine species Miamensis avidus in the whole range of
existence investigated (Kaneshiro et al., 19б9). The similar data were obtained by other researcher for amoeboid species Chaos carolinense (Riddic, 19б8, as Pelomyxa carolinensis). Apparently, it is possible to assume that the internal medium of the protists cell is necessarily hyperosmotic in relation to the environment.
The identical form of described breakpoint regression curve is characteristic of freshwater species P. caudatum and considered to be euryhaline P. nephridiatum. It is necessary to assume that the greater range of potential salinity tolerance in P. nephridiatum is connected with the better osmoregulational abilities. Such improvement of available already reached mechanism intensification ensured hyperosmolarity of internal medium and is identical in freshwater and euryhaline paramecia.
Boundaries of ciliates freshwater species distribution are limited by critical salinity of 5-8 %% (Smurov and Fokin, 1999). The area of critical salinity values approximately coincides with values specified by Beadle (1959). Such species as P. bursaria, P. caudatum have breakpoint on the diagram within the limits of 0.5-3 % along the axis of acclimation salinity and within the limits of 3-7 % along the axis of test salinity. It is possible to assume, that in the point, where extreme of limit is achieved, further hyperosmotic regulation is impossible. Hence, the species having breakpoint in the specified limits should be named freshwater species. Diagrams of their potential salinity tolerance have growth of the upper limit value approximately up to salinity of acclimation value 3 %o. P sexaurelia is capable to exist at higher salinities - up to 15 %o, but the breakpoint of the curve of tolerant limit is at 5.5 %o. For P. nephridiatum the value of breakpoint is equal to 4.5 %o, and it insignificantly differs from characteristic of species of freshwater origin. Beside that, this species is capable to exist at salinities considerably exceeding the critical one. There is a question, whether the extreme of the diagram of tolerant salinity limit is the certificate of origin from freshwater ancestor?
Existence of species and stocks which can adapt themselves in wide and narrow ranges of test salinity scale allows to elaborate recommendations for their experimental study. To estimate salinity tolerant range of paramecia having mortality of the type 2 it is suitable to use the method of stock preservation (Smurov and Fokin, 1998). This method allows estimating tolerant range quickly and precisely. If curve of mortality belongs to the type 1, the data estimation by resistance can be more accurate and quick.
Acknowledgments
I am grateful to Dr. S.I.Fokin, Dr. I.S.Plotnikov, Dr. A.A.Filippov and A.A.Kudryavtsev for useful comments.
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Address for correspondence: Alexei O. Smurov. Zoological Institute RAS, Universitetskaya nab. 1, St. Petersburg, 199034, Russia. E-mail: aral@zin.ru.
The manuscript is presented by S.I.Fokin
