Hypo-osmotic or Ca2+-rich external conditions trigger extra contractile vacuole complex generation in Paramecium multimicronucleatum
Pacific Biomedical Research Center, Snyder Hall 306, University of Hawaii at Manoa, 2538 The Mall, Honolulu, HI 96822, USA
* Author for correspondence (e-mail: naitoh{at}pbrc.hawaii.edu)
Accepted 1 September 2003
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Summary |
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Key words: contractile vacuole complex, osmoregulation, Ca2+ regulation, organelle biogenesis, Paramecium multimicronucleatum
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Introduction |
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Many species of Paramecium possess two CVCs in a single cell.
Individual cells in a population, however, have been reported to possess three
or more CVCs, i.e. P. multimicronucleatum
(Powers and Mitchell, 1910;
King, 1935
;
Allen et al., 1990
), P.
caudatum (Bhatia, 1923
;
Wichterman, 1946
), P.
aurelia complex (King,
1954
) P. ugandae
(Wichterman, 1986
). What
factor(s) causes generation of extra CVCs has not been determined.
In the present study, we demonstrate that in P. multimicronucleatum generation of extra CVCs is enhanced by exposing the cells to either lowered osmolarity or tincreased Ca2+ concentration. Furthermore, we found that generation of extra CVCs under these conditions was controlled by a mechanism that differs from the mechanism that controls normal duplication of CVCs during cell division.
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Materials and methods |
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Experimental solutions
The standard saline solution for adaptation was a mixture containing (mmol
l-1 final concentration) 2.0 KCl, 0.25 CaCl2 and 1.0
Mops-KOH (pH 7.0), which had an osmolarity of 4 mosmol l-1
(Naitoh et al., 1997).
Osmolarities of some experimental solutions were varied by adding sorbitol to
the standard saline solution. In some experiments, concentrations of KCl
and/or CaCl2 were varied. The osmolarity of each solution was
determined by using a freezing-point osmometer. A stock solution of
aphidicolin (Sigma, St Louis, MO, USA) dissolved in dimethyl sulphoxide (DMSO)
at 25 mmol l-1 was added to the cell suspension to inhibit cell
cycle progression.
Immunofluorescence microscopy of the CVC
For counting the number of CVCs in Paramecium, cells were fixed
for 30 min in formaldehyde (3% in 50 mmol l-1 phosphate buffer, pH
7.4) and permeabilized for 20 min in cold (-20°C) acetone before treatment
with a monoclonal antibody (mAb) raised against the smooth spongiome (SS-1
mAb, IgM) (Ishida et al.,
1996). This was followed by treatment with
fluorescein-isothiocyanate (FITC)-conjugated rabbit anti-mouse IgM. The cells
were observed by using a Zeiss microscope equipped with epifluorescence
illumination and a filter appropriate for FITC (B-2E, Nikon, Tokyo, Japan).
Images of the cells were taken on Kodak Tri-X film. The number of CVCs per
cell (NCVC), in more than 300 cells, was determined for
each experiment.
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Results |
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Frequency distribution of the number of radial arms in a single CVC
(NRA) in cells growing in axenic culture medium compared to cells
adapted to a 4 mosmol l-1 standard saline solution
CVCs of cells adapted to a 4 mosmol l-1 standard saline solution
for 24 h differed from those growing in axenic culture medium not only in
NCVC but also in the number of radial arms in a single CVC
(NRA). NRA was larger in cells adapted
to the saline solution than those in the axenic culture medium.
Fig. 2 shows the frequency
distributions of NRA for the two groups of cells, growing
in the culture medium (black columns) or adapted to the standard saline
solution (white columns). The mean NRA for cells adapted
to the standard saline solution was 11.2±2.2 (N=135) and was
significantly (t-test, P<10-18) larger than
that for the cells in the axenic culture medium (9.0±1.5;
N=141). Moreover, NRA exhibited a wider
distribution (variance s=4.8) in cells adapted to the saline solution than in
cells in the axenic culture medium (s=2.3). In addition, the radial arms of
the cells adapted to the standard saline solution frequently exhibited
branching (Fig. 1B2; white
arrowhead).
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Change in the number of cells with extra CVCs after transferring the
cells into saline solutions that have different osmolarities from that of the
axenic culture medium
The time course of change in the number of cells that possess three or more
CVCs (the cells with extra CVCs) was determined after transferring the cells
from their culture medium to one of three saline solutions with different
osmolarities 4, 64 or 144 mosmol l-1, respectively.
Fig. 3 shows the number of
cells with extra CVCs as a function of time after the transfer. The number
increased with time in all cases, even when the osmolarity was higher than the
axenic culture medium (84 mosmol l-1). The increase was the highest
in the solution with the lowest osmolarity, i.e. 4 mosmol l-1.
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Change in the number of cells with extra CVCs after transferring the
cells into saline solutions with different K+ or Ca2+
concentrations
In order to investigate external factors other than the osmotic factor that
affect the extra CVC generation, the time course of change in the number of
cells with extra CVCs was determined after transferring the cells from their
culture medium to several different saline solutions with (1) a varied
K+ concentration (0.1, 2.0 or 5.0 mmol l-1) at a
constant Ca2+ concentration (0.25 mmol l-1) or (2) a
varied Ca2+ concentrations (0.001, 0.1, 0.25 or 1.0 mmol
l-1) at a constant K+ concentration (2.0 mmol
l-1). The osmolarity of each saline solution was kept constant at
84 mosmol l-1, which equalled the osmolarity of the axenic culture
medium. These cells were, therefore, not subjected to a change in the
osmolarity upon their transfer into any of the saline solutions.
As is shown in Fig. 4A, the time course for the cells in each of three different solutions having different K+ concentrations was essentially identical with each other. The number increased with time from its initial value of approximately 12% to approximately 40% at 48 h.
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Fig. 4B shows the time course for the cells in each of four different solutions with different Ca2+ concentrations. In each case the number increased with time and reached its plateau value at around 48 h. The plateau value was larger as the Ca2+ concentration increased.
Change in the number of cells with extra CVCs after returning cells
to axenic culture medium
In order to determine how the number of cells with extra CVCs changes after
the cells are returned to the culture medium from a saline adaptation
solution, a sample of cells obtained from the axenic culture was first
transferred into a 4 mosmol l-1 saline solution and kept immersed
in this solution for 18 h. The cells were then returned to fresh axenic
culture medium and kept immersed in this solution for 60 h. Thenumber of cells
that possess extra CVCs was determined for cells obtained from the sample at
intervals of time during this 60 h. The cell density was also determined at
the same time and presented as a percentage of the density of cells at -18 h,
corresponding to the time when the cells were transferred into the 4 mosmol
l-1 saline solution.
As shown in Fig. 5, the number of cells with extra CVCs (solid circles) that had increased to approximately 45% during the adaptation of the cells to the saline solution (from -18 to 0 h) did not change during the first 12 h after returning the cells to the axenic culture medium. The cell density (Fig. 5, open circles) was also unchanged during this same period. The number began to decrease between 12 and 24 h and continued to decrease. By 60 h the number had returned to its initial value of approximately 12%. The cell density had begun to increase by 24 h and continued to increase to the end of the 60 hexperiment.
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Locations in cells where extra CVCs are generated
In a normal cell division cycle, each `daughter' CVC will be formed
anterior to a pre-existing `mother' CVC immediately before the start of cell
division (King, 1935;
Allen et al., 1990
).
Paramecium cells, therefore, typically have four CVCs just before
cytokinesis occurs (Fig.
6A).
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On the other hand, as described above, under a condition of either lowered osmolarity or higher Ca2+ concentration or both, some non-dividing cells formed one extra CVC at either one of two different locations. Firstly, a new CVC could be generated in the posterior region of the cell posterior to the posterior CVC. This newly generated CVC frequently seemed to be connected to a long radial arm of the mature posterior CVC (Fig. 6B). Secondly, a new CVC could also be generated by the apparent binary division of the anterior CVC (Fig. 6C). The extra CVC formed by binary division was similar to its mother CVC in both size and shape.
Effects of aphidicolin on the extra CVC generation
To determine whether extra CVC generation is related to germinal DNA
synthesis in the micronuclei, the effect of aphidicolin, a potent inhibitor of
DNA synthesis in the micronuclei of Paramecium
(Sabaneyeva et al., 1999), as
well as in the nuclei of other eukaryotic cells, on extra CVC generation was
examined. Two groups of cells obtained from the axenic culture were
transferred, one into a 4 mosmol l-1 standard saline solution and
the other into the same saline solution containing 25 µmol l-1
aphidicolin. The numbers of cells that had 1, 2, 3, 4 or 5 or more CVCs,
respectively, were determined for these two cell groups after they had been
adapted to the respective saline solutions for 18 h. As is clearly shown in
Fig. 7, the frequency
distribution of cells with different NCVC was identical
between these two cell groups.
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Discussion |
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The number of cells with extra CVCs increased when the cells were transferred into a series of saline solutions with decreasing osmolarities but in which the ionic compositions remained the same (Fig. 3). This implies that lowering the external osmolarity is one factor that influences the extra CVC generation.
However, it was also observed that the number of cells with extra CVCs also increased slightly in a 144 mosmol l-1 saline solution (Fig. 3). This osmolarity is higher than the osmolarity of the culture medium (84 mosmol l-1), and the result implies that external factors other than lowering the osmolarity can also influence the mechanism for generation of extra CVCs.
The time course required for a population of cells to acquire extra CVCs after transfer from an 84 mosmol l-1 axenic culture medium into saline solutions also at 84 mosmol l-1, but with different concentrations of K+ (0.1, 2.0 or 5.0 mmol l-1) or Ca2+ (0.001, 0.1, 0.25 or 1.0 mmol l-1), clearly demonstrated that generation of extra CVCs was essentially unaffected by varying the external K+ concentration (Fig. 4A), while it was enhanced by increasing the external Ca2+ concentration (Fig. 4B). This implies that an increase in the external Ca2+ concentration is a second factor that promotes the extra CVC generation. This hypothesis may account for the increase in the number of cells with extra CVCs found in 144 mosmol l-1 saline solutions (Fig. 3; filled circles) which, in fact, did have higher Ca2+ concentrations than the axenic culture medium, since the free Ca2+ concentration in the axenic culture medium we used was less than 0.05 mmol l-1 (M. Iwamoto, R. D. Allen and Y. Naitoh, unpublished preliminary data) while that in the standard saline solution was 0.25 mmol l-1.
It was also expected that more Ca2+ ions would enter the cell
through Ca2+ channels in the cell surface membrane when the cells
were in the standard saline solution than in the axenic culture medium.
Voltage-dependent Ca2+ channels
(Naitoh and Eckert, 1968;
Naitoh, 1979
;
Ehrlich et al., 1984
;
Machemer, 1988
) have been
reported to be present in the ciliary membrane of Paramecium, while
mechano-sensitive Ca2+ channels
(Naitoh and Eckert, 1969
) are
found in the somatic membrane (Dunlap,
1977
; Ogura and Takahashi,
1976
; Ogura and Machemer,
1980
). Elimination of excess Ca2+ from the cytosol is
extremely important for the cell to be able to maintain its
Ca2+-mediated intracellular signaling systems. The cytosolic free
Ca2+ concentration in the Paramecium cell was assumed to
be around 10-8 mol l-1 by Naitoh and Kaneko
(1972
). More recently
Plattner's group determined the cytosolic Ca2+ concentration of
Paramecium to be 5-8x10-8 mol l-1, using
a fluorochrome analysis method (Klauke and
Plattner, 1998
; Plattner and
Klauke, 2001
). Many cells have Ca2+-pumps
(Ca2+-ATPases) in their plasma membranes to eliminate excess
cytosolic free Ca2+ ions (Edes
and Kranias, 2001
). In P. multimicronucleatum a
Ca2+-pump has not yet been identified in the plasma membrane;
however, we recently found that the CVC of P. multimicronucleatum can
accumulate as much as 20 mmol l-1 Ca2+
(Stock et al., 2002b
). Such a
high concentration of Ca2+ in the CVC suggests that the CVC plays
an important functional role in segregating excess cytosolic Ca2+
and subsequently eliminating it from the cell. To achieve Ca2+
regulation we now find that the Paramecium cell can also generate
extra CVCs under conditions of high external Ca2+
concentration.
Ca2+-pumps have been reported in the CV membrane of
Dictyostelium, and have a high homology to other P-type pumps
(Moniakis et al., 1995), so
that Ca2+ transport into the CV by this pump may require a proton
gradient between the cytosol and the inside of the CV
(Moniakis et al., 1999
). We
have demonstrated that numerous proton pumps are indeed present in CV
membranes of Paramecium (Fok et
al., 1995
). Heuser et al.
(1993
) demonstrated V-ATPases
are also found in the CV membrane of Dictyostelium. Thus CVCs in
Dictyostelium may also play a role not only in osmoregulation but
also in Ca2+ discharge from the cytoplasm. In fact V-ATPases seem
to be a constant component of contractile vacuole systems as they have also
been shown in the tiny CVs of Phytophthora zoospores
(Mitchell and Hardham, 1999
).
We now need to see if there is a functional link between proton pumps and
Ca2+ uptake in the CVCs of Paramecium.
Another finding is that the number of radial arms in a single CVC (NRA) is larger in cells adapted to a 4 mosmol l-1 standard saline solution than those in cells in the axenic culture medium (Fig. 2). It can be assumed that an increase in NRA will enhance the rates of segregation of both excess cytosolic water and Ca2+ in a cell in addition to an increased NCVC.
Cell division normalizes NCVC
As is clearly shown in Fig.
5, the number of cells with extra CVCs that increased during
adaptation of the cells to a 4 mosmol l-1 saline solution started
to decrease to its normal value (approximately 12%) as the cells began to
multiply, approximately 24 h after the cells were returned to a normal axenic
culture medium. This implicates cell division in the reduction of
NCVC. It can, therefore, be expected that
NCVC might increase, if cell division is inhibited. In
fact, a slight increase in the number of cells with extra CVCs was observed in
an 84 mosmol l-1 saline solution containing a very low
Ca2+ concentration (0.001 mmol l-1;
Fig. 4B, filled triangles).
Upon transferring into this solution, the cells encountered neither a lowering
of the external osmolarity nor an increase in the external Ca2+
concentration, but they did encounter a deletion of the external nutrients
that are necessary for cell division (Fig.
5; open circles).
Extra CVC generation is governed by a mechanism(s) that differs from
duplication of CVCs during normal cell division
We clearly demonstrated that extra CVC generation was enhanced by
transferring the cells into hypo-osmotic or Ca2+-rich saline
solution (Figs 1,
3,
4) but in which normal cell
division stopped (Fig. 5). It
is known that during normal cell division, the appearance of new daughter CVCs
is followed immediately by the first signs of the formation of a division zone
across the middle of the cell (Fig.
6A) (Allen et al.,
1990; Fok et al.,
2002
). It is therefore suggested that extra CVC generation is
independent of cell cycle progression. In fact, addition of aphidicolin, which
inhibits micronuclear DNA synthesis in Paramecium
(Sabaneyeva et al., 1999
), had
no effect on the generation of extra CVCs
(Fig. 7). This supports the
hypothesis that extra CVC generation is independent of micronuclear DNA
synthesis on which normal CVC duplication cycles depend.
In addition, the manner in which extra CVC generation occurs differs
morphologically from that of daughter CVC generation during normal cell
division. The two daughter CVCs always arise anterior to their mother CVCs
(King, 1935;
Kaneda and Hanson, 1974
;
Allen et al., 1990
) at nearly
the same time in the cell cycle, so that cells showing signs of a division
furrow almost always have four CVCs (Fig.
6A). On the other hand, in many cells one extra CVC is generated
posterior to the posterior CVC (Fig.
6B) or an extra CVC can be generated by the division of the
anterior CVC (Fig. 6C). These
morphological differences also strongly support the idea that the mechanism(s)
that governs the generation of the extra CVCs differs from that which governs
generation of new CVCs during cell division.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Allen, R. D. and Fok, A. K. (1988). Membrane dynamics of the contractile vacuole of Paramecium. J. Protozool. 35,63 -71.
Allen, R. D., Ueno, M. S., Pollard, L. W. and Fok, A. K. (1990). Monoclonal antibody study of the decorated spongiome of contractile vacuole complexes of Paramecium. J. Cell Sci. 96,469 -475.[Abstract]
Bhatia, B. L. (1923). On the significance of extra contractile vacuoles in Paramecium caudatum. J. R. Microsc. Soc. 262,69 -72.
Dunham, P. B. and Kropp, D. L. (1973). Regulation of solute and water in Tetrahymena. In Biology of Tetrahymena (ed. A. M. Elliot), pp.165 -198. Dowden: Hutchinson & Ross.
Dunlap, K. (1977). Localization of calcium channels in Paramecium caudatum. J. Physiol. 271,119 -133.[Abstract]
Edes, I. and Kranias, E. G. (2001). Ca2+-ATPases. In Cell Physiology Source Book (third edition) (ed. N. Sperelakis), pp 271-282. San Diego, San Francisco, New York, Boston, London, Sydney, Tokyo: Academic Press.
Ehrlich, B. E., Finkelstein, A., Forte, M. and Kung, C. (1984). Voltage-dependent calcium channels from Paramecium cilia incorporated into planar lipid bilayers. Science 225,427 -428.[Medline]
Fok, A. K. and Allen, R. D. (1979). Axenic Paramecium caudatum. I. Mass culture and structure. J. Protozool. 26,463 -470.[Medline]
Fok, A. K., Aihara, M. S., Ishida, M., Nolta, K. V., Steck, T.
L. and Allen, R. D. (1995). The pegs on the decorated tubules
of the contractile vacuole complex of Paramecium are proton pumps.
J. Cell Sci. 108,3163
-3170.
Fok, A. K., Yamauchi, K., Ishihara, A., Aihara, M. S., Ishida, M. and Allen, R. D. (2002). The vacuolar-ATPase of Paramecium multimicronucleatum: gene structure of the B subunit and the dynamics of the V-ATPase-rich osmoregulatory membranes. J. Eukar. Microbiol. 49,185 -196.[Medline]
Heuser, J., Zhu, Q. and Clarke, M. (1993). Proton pumps populate the contractile vacuoles of Dictyostelium amoebae. J. Cell Biol. 121,1311 -1327.[Abstract]
Ishida, M., Aihara, M. S., Allen, R. D. and Fok, A. K.
(1993). Osmoregulation in Paramecium: the locus of fluid
segregation in the contractile vacuole complex. J. Cell
Sci. 106,693
-702.
Ishida, M., Fok, A. K., Aihara, M. S. and Allen, R. D.
(1996). Hyperosmotic stress leads to reversible dissociation of
the proton pump-bearing tubules from the contractile vacuole complex in
Paramecium. J. Cell Sci.
109,229
-237.
Kaneda, M. and Hanson, E. D. (1974). Growth patterns and morphogenetic events in the cell cycle of Paramecium aurelia. In Paramecium, A Current Survey (ed. W. J. van Wagtendonk), pp. 219-262. Amsterdam, London, New York: Elsevier.
King, R. L. (1935). The contractile vacuole of Paramecium multimicronucleata. J. Morphol. 58,555 -571.
King, R. L. (1954). Origin and morphogenetic movement of the pores of the contractile vacuoles in Paramecium aurelia.J. Protozool. 1,121 -130.
Kitching, J. A. (1956). Contractile vacuole of protozoa. In Protoplasmatologica, vol.III (ed. L. V. Heilbrunn and F. Weber), pp.1 -45. Wien: Springer-Verlag.
Klauke, N. and Plattner, H. (1998). Caffeine-induced Ca2+ transients and exocytosis in Paramecium cells. A correlated Ca2+ imaging and quenched-flow/freeze-fracture analysis. J. Membr. Biol. 161,65 -81.[CrossRef][Medline]
Machemer, H. (1988). Electrophysiology. In Paramecium (ed. H.-D. Görtz), pp.185 -215. Berlin, Heidelberg, New York, London, Paris, Tokyo: Springer-Verlag.
Mitchell, H. J. and Hardham, A. R. (1999). Characterisation of the water expulsion vacuole in Phytophthora nicotianae zoospores. Protoplasma 206,118 -130.
Moniakis, J., Coukell, M. B. and Forer, A.
(1995). Molecular cloning of an intracellular P-type ATPase from
Dictyostelium that is up-regulated in calcium-adapted cells.
J. Biol. Chem. 270,28276
-28281.
Moniakis, J., Coukell, M. B. and Janiec, A.
(1999). Involvement of the Ca2+-ATPase PAT1 and the
contractile vacuole in calcium regulation in Dictyostelium discoideum.J. Cell Sci. 112,405
-414.
Naitoh, Y. (1979). Membrane currents in voltage-clamped Paramecium and their relations to ciliary motion. Acta Protozool. 18,1 -6.
Naitoh, Y. and Eckert, R. (1968). Electrical properties of Paramecium caudatum: Modification by bound and free cations. Z. Vergl. Physiol. 61,427 -452.
Naitoh, Y. and Eckert, R. (1969). Ionic mechanisms controlling behavioral responses in Paramecium to mechanical stimulation. Science 164,963 -965.[Medline]
Naitoh, Y. and Kaneko, H. (1972). Reactivated triton-extracted models of Paramecium: Modification of ciliary movement by calcium ions. Science 176,523 -524.[Medline]
Naitoh, Y., Tominaga, T., Ishida, M., Fok, A. K., Aihara, M. S.
and Allen, R. D. (1997). How does the contractile vacuole of
Paramecium multimicronucleatum expel fluid? Modelling the expulsion
mechanism. J. Exp. Biol.
200,713
-721.
Ogura, A. and Machemer, H. (1980). Distribution of mechanoreceptor channels in the Paramecium surface membrane. J. Comp. Physiol. 135,233 -242.
Ogura, A. and Takahashi, K. (1976). Artificial deciliation causes loss of calcium-dependent responses in Paramecium.Nature 264,170 -172.[Medline]
Plattner, H. and Klauke, N. (2001). Calcium in ciliated protozoa: sources, regulation, and calcium-regulated cell functions. Int. Rev. Cytol. 201,115 -208.[Medline]
Powers, J. H. and Mitchell, C. (1910). A new species of Paramecium (Paramecium multimicronucleata) experimentally determined. Biol. Bull. 19,324 -332.
Sabaneyeva, E., Tao, W. and Verbelen, J. P. (1999). Aphidicolin inhibits DNA replication in the micronucleus and blocks cytokinesis in Paramecium caudatum. Cell Biol. Int. 23,859 -862.[CrossRef][Medline]
Stock, C., Allen, R. D. and Naitoh, Y. (2001).
How external osmolarity affects the activity of the contractile vacuole
complex, the cytosolic osmolarity and the water permeability of the plasma
membrane in Paramecium multimicronucleatum. J. Exp.
Biol. 204,291
-304.
Stock, C., Grønlien, H. K., Allen, R. D. and Naitoh,
Y. (2002a). Osmoregulation in Paramecium: in situ
ion gradients permit water to cascade through the cytosol to the contractile
vacuole. J. Cell Sci.
115,2339
-2348.
Stock, C., Grønlien, H. K. and Allen, R. D. (2002b). The ionic composition of the contractile vacuole fluid of Paramecium mirrors ion transport across the plasma membrane. Eur. J. Cell Biol. 81,505 -515.[Medline]
Wichterman, R. (1946). Unstable micronuclear behavior in an unusual race of Paramecium. Anat. Rec. 94, 94.
Wichterman, R. (1986). Classification and Species of Paramecium. In The Biology of Paramecium (second edition), pp. 1-59. New York, London: Plenum Press.