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Cell proliferation of Paramecium tetraurelia on a slow rotating clinostat
Advances in Space Research 39 (2007) 1166–1170 www.elsevier.com/locate/asr
Cell proliferation of Paramecium tetraurelia on a slow rotating clinostat Satoe Sawai a
a,*
, Yoshihiro Mogami b, Shoji A. Baba
a
Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka 2-1-1, Tokyo 112-8610, Japan b Department of Biology, Ochanomizu University, Otsuka 2-1-1, Tokyo 112-8610, Japan Received 26 September 2006; received in revised form 6 January 2007; accepted 7 February 2007
Abstract Paramecium is known to proliferate faster under microgravity conditions, and slower under hypergravity. Experiments using axenic culture medium have demonstrated that hypergravity affected directly on the proliferation of Paramecium itself. In order to assess the mechanisms underlying the physiological effects of gravity on cell proliferation, Paramecium tetraurelia was grown under clinorotation (2.5 rpm) and the time course of the proliferation was investigated in detail on the basis of the logistic analysis. On the basis of the mechanical properties of Paramecium, this slow rate of the rotation appears to be enough to simulate microgravity in terms of the randomization of the cell orientation with respect to gravity. P. tetraurelia was cultivated in a closed chamber in which cells were confined without air bubbles, reducing the shear forces and turbulences under clinorotation. The chamber is made of quartz and silicone rubber film; the former is for the optically-flat walls for the measurement of cell density by means of a non-invasive laser optical-slice method, and the latter for gas exchange. Because of the small dimension for culture space, Paramecium does not accumulate at the top of the chamber in spite of its known negative gravitactic behavior. We measured the cell density at regular time intervals without breaking the configuration of the chamber, and analyzed the proliferation parameters by fitting the data to a logistic equation. As a result, P. tetraurelia showed reduced proliferation under slow clinorotation. The saturation of the cell density as well as the maximum proliferation rate decreased, although we found no significant changes on the half maximal time for proliferation. We also found that the mean swimming velocity decreased under slow clinorotation. These results were not consistent with those under microgravity and fast rotating clinostat. This may suggest that randomization of the cell orientation performed by slow rotating clinostat has not the same effect on Paramecium as that under microgravity that may affect the proliferation as the result of the reduced cost of propulsion. 2007 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: Paramecium; Clinorotation; Simulated microgravity; Proliferation rate; Swimming velocity
1. Introduction Previous space experiments aboard the Soviet orbital station Salyut 6 (CYTOS experiment) and the Space Shuttle (D1 mission) reported that the proliferation of Paramecium became faster (about 160%) under microgravity in space (Planel et al., 1981; Richoilley et al., 1986). The same authors reported that proliferation became slower (about 70%) under hypergravity (20g) provided by centrifugation (Tixador et al., 1984; Planel et al., 1990; Richoilley et al.,
*
Corresponding author. E-mail address: [email protected] (S. Sawai).
1993). Experiments using axenic culture medium have demonstrated the direct effect of hypergravity on the proliferation of Paramecium itself, other than the indirect effect on the proliferation of nutrient bacteria grown in non-axenic culture (Richoilley et al., 1993; Kato et al., 2003). Mechanisms underlying the gravity dependent changes in proliferation could be explained in terms of the energetics of the proliferation as well as of the motile activities of the cell. Because Paramecium modulate their propulsive thrust depending on the orientation of the cell body with respect to the gravity vector, i.e. increasing propulsive thrust in upward swimming and decreasing it in downward (Machemer et al., 1991; Ooya et al., 1992). This gravityinduced change in propulsion, gravikinesis, is explained
0273-1177/$30 2007 COSPAR. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.asr.2007.02.023
S. Sawai et al. / Advances in Space Research 39 (2007) 1166–1170
on the basis of cellular mechanosensitivity in combination with close coupling between the membrane potential and ciliary locomotors activity (Machemer, 1990). It seems likely that Paramecium consumes much energy when swimming upwards than that when swimming downwards. Mogami et al. (2001) demonstrated that the gravitactic orientation of Paramecium is mechanically biased by the torque mainly due to the fore-aft asymmetry of the cell body (gravitaxis). The upward-orienting torque increases with increase in gravity acceleration, so that the fraction of the upward-orienting cell would decrease under reduced gravity, and increase under hypergravity. This would result in a change in energy expenditure for propulsion under different gravity accelerations. Thrust force would be increased under hypergravity and hence reduced under microgravity. If the energy available for physiological events of the cell is limited, changes in the energy for propulsion may lead to the changes in the energy stock for the proliferation. The larger stock of energy under microgravity as the result of the reduced propulsive work might enhance the proliferation activity and smaller stock would reduce this activity under hypergravity. In order to assess the feasibility of the assumption above, Paramecium tetraurelia was grown under simulated microgravity performed by clinorotation. If it is the case, we may expect the cell proliferation of Paramecium to be enhanced by randomization of the cell orientation by means of clinorotation. 2. Materials and methods P. tetraurelia was cultivated axenically as described previously (Kato et al., 2003) in the medium of Soldo et al. (1966) with the modifications by Fok and Allen (1979). We used a closed chamber for the cell culture under clinorotation in which cells were confined without air bubbles for reducing the shear forces and turbulences under rotation (Fig. 1). The chamber had a culture space with an inner dimension of 3 · 3 · 60 mm, which had an inlet and outlet for the medium. Three walls of the culture space were made of quartz and the rest of silicone rubber film.
a
Moisture space
1167
The quartz walls provided the optically-flat windows for the measurement of cell density by means of a non-invasive laser optical-slice method (Mogami et al., 2000; Kato et al., 2003). Gas exchange for respiration was carried out through the silicone rubber film between the medium and the moist air in the space placed next to the culture space. This prevented the formation of air bubbles in the culture medium caused by the evaporation of water in association with gas exchange. The depth of the culture volume was restricted to 3 mm when the chamber was placed horizontally. In this shallow space, Paramecium showed roughly even vertical distribution rather than the apparent accumulation at the top of the culture volume as observed in a deep water column. The culture chamber was placed on a rotator (RT-5 TAITEC, Tokyo) with the long axis of the chamber kept horizontal and rotated around the long axis (clinorotation) or around the short axis perpendicular to the long axis (control rotation) (Fig. 2). Both the direction and the speed of rotation (2.5 rpm) were kept constant throughout an experiment. For the horizontal rotation, the chamber was placed with the silicone rubber as the side wall of the culture space. Cell density was measured without breaking the configuration of the chamber by optical-slice method (Kato et al., 2003) with removing the chamber from the rotators. The time course of the cell proliferation under clinorotation was analyzed on the basis of the logistic growth equation. According to the three parameters determined by means of the least squares fitting of the equation to the experimental data as described in Kato et al. (2003), the kinetic parameter of proliferation (saturation cell density, maximum pro-
Culture space
Silicone rubber film
b
Moisture space Silicone rubber film Culture space
Fig. 1. Schematic drawings of the closed chamber for the culture of Paramecium under clinorotation. Side view (a) and front view (b) of the chamber showing the culture space and moist air space for gas exchange.
Fig. 2. (a) An overview of the rotator developed for clinorotation. Arrow indicates one of the closed chambers. (b) Schematic drawing of the rotation of the culture space in the clinorotation and the control horizontal rotation.
S. Sawai et al. / Advances in Space Research 39 (2007) 1166–1170
3. Results
ð1Þ
where N is the cell density as a function of time, t, and K, a and l are the parameters characteristic of the individual culture. Eq. (1) is derived from a differential equation of dN =dt ¼ lN ð1 N =KÞ;
ð2Þ
where the growth rate is defined by the product of a proportional constant (l, intrinsic growth rate) and the terms one of which is related to the cell density in culture (N) and another to the density dependent inhibitory effect (1 N/ K); e.g., the reduction in available resources. According to the least squares fitting of parameters in Eq. (1) to the experimental data, the kinetic parameters of proliferation, i.e. saturation (maximal) cell density, maximum proliferation rate and half maximal time (the time for half maximal cell density) were calculated as K, lK/4 and (ln a)/l, respectively. As shown in Fig. 4, clinorotation reduced the saturation cell density and the maximum proliferation rate, although we found no significant changes on half maximal time. Similar reduction of the saturation density and the maximum growth rate was also found under hypergravity condition (Kato et al., 2003). However, reduction by hypergravity was associated with the increase in half maximal time. This is a remarkable difference between the inhibitory effects provided by hypergravity and clinorotation. Effects of clinorotation on Paramecium were also found in propulsive activity. The mean swimming velocity of P. tetraurelia in the logarithmic phase of the proliferation under slow rotating clinostat (2.5 rpm) decreased in average to 85.0 ± 4.7% of the control (data from three duplicated or triplicated measurements containing the measurements on >150 individual cells, P < 0.05). The result was not consistent with the hypergravity experiment: P. tetraurelia has higher swimming activity under hypergravity (Kato, 2004).
0.2
P = 0.60
0.15 0.1 0.05 0
30000
400
P < 0.001
300 200 100 0
25000 15000 10000 5000 0
0
24
48 72 time (hrs)
96
Fig. 3. The time course of the cell proliferation under clinorotation (2.5 rpm, filled circle) as well as control horizontal rotation of the same angular velocity (open circle). Means ± SD of seven duplicated or triplicated measurements are shown. Curves are the result of the least squares fitting of the logistic growth equation.
800 600 400 200 0
P = 0.07
P = 0.05 half maximal time (hr)
20000
maximum proliferation rate (cells/ml hr)
cell density (cells/ml)
As reported briefly (Mogami et al., 2000), Paramecium proliferated in a similar manner in the closed culture chamber to that observed in the open surface culture. Maximal cell density found in the steady state was 2–3 · 104 cell/ml, which is similar to the maximal value in the open surface culture (Kato et al., 2003). This means that the closed culture system used in this study is well-suited to the experiment on the proliferation of Paramecium performed under good physiological conditions. Similar profiles of the proliferation were kept in the culture under horizontal rotation; maximal cell density in the steady state with and without horizontal rotation (2.5 rpm), was 2.83 ± 0.82 · 104 (cells/ml, mean ± SD, n = 6) and 2.30 ± 0.82 · 104 (n = 5), respectively (P = 0.3). Since we did not find any difference in the proliferation under horizontal rotation at various angular velocities (0.2–4.8 rpm), we used the culture under horizontal rotation as the control to the culture under clinorotation. Fig. 3 shows the time course of the cell proliferation under slow rotating clinostat (2.5 rpm) as well as under control horizontal rotation. Cell proliferation was reduced under clinorotation. In order to quantitatively assess the reduction, the time course of the cell proliferation under clinorotation and control horizontal rotation were analyzed on the basis of the logistic growth equation
N ðtÞ ¼ K=f1 þ a expðltÞg;
saturation cell density ( 102 cells/ml)
liferation rate and half maximal time) were calculated. Temperature during culture was regulated at 24 C. Swimming speed of P. tetraurelia was measured from the video recordings under a dark-field illumination performed by the laser optical slice. For this purpose, culture space was illuminated vertically by a slit-laser, and cells swimming in any direction within a vertical illumination slit were used for the measurement. Positions of individual cells were determined by a laboratory-made computer assisted tracking software (Shiba et al., 2005), and the average velocity was calculated from the changes in the distances at defined time intervals. Probability of statistical significance (P) was determined using Student’s t-test.
intrinsic growth rate (/hr)
1168
400 300 200 100 0
Fig. 4. The kinetic parameters of proliferation of P. tetraurelia cultivated under clinorotation (2.5 rpm, filled bar) as well as control horizontal rotation (open bar). Means ± SD obtained from the least squares fitting of Eq. (1) to the seven sets of experiments.
S. Sawai et al. / Advances in Space Research 39 (2007) 1166–1170
The differences found in the profiles of the proliferation as well as the propulsive activity suggest that slow clinorotation caused the inhibitory effect of the proliferation of Paramecium depending on the different mechanisms from those working under hypergravity. 4. Discussion Clinorotation has been known to simulate microgravity in space. However, the clinorotation used in the present paper (slow clinorotation) did not confirm the increased proliferation rate of Paramecium in real microgravity (Planel et al., 1981) and on a fast rotation clinostat (>60 rpm) (Ayed et al., 1992; Hemmersbach-Krause et al., 1990). For the gravitactic behavior of Paramecium, clinorotation may affect both the vertical distribution of the whole population and the upward orientation of individual cells. We used closed culture space with a depth of 3 mm, which is much less than the scale depth of the gravitactic distribution of free-swimming Paramecium (Roberts, 1970). As the results, cells were observed to almost evenly distribute vertically as well as horizontally in the culture space placed without rotation. Therefore, clinorotation might not affect any more to change the distribution profile. According to the theoretical studies on the gravitactic orientation of swimming microorganisms in the rotating fluid field (Kessler, 1986; Kessler et al., 1998), organisms tumble with the fluid when the angular velocity of the fluid rotation is greater than the gravity-dependent orientation rate of the organisms. By the analysis of the sedimentation of immobilized Paramecium, the maximum rate of the orientation was measured with 0.08 rad/s (Mogami et al., 2001), which is much smaller than the angular velocity of clinorotation (2.5 rpm = 0.26 rad/s). It is therefore likely that Paramecium oriented randomly with respect to the gravity vector as the result of the clinorotation at this angular velocity. These indicate that the slow clinorotation used in these studies was effective for randomizing the orientation of the cell with keeping the homogenous vertical distribution in the narrow chamber. Ayed et al. (1992) reported a stimulating effect of clinorotation on the proliferation of Paramecium. The effect depends on rotation rate, and they reported about 25% increase in the cell density at the higher angular velocity (80–90 rpm). We confirmed the inhibitory effect of clinorotation up to 4.8 rpm in the configuration used in this study, although the possibility would not excluded that the rotation at higher angular velocity has stimulatory effects. The other possibility would be considered that the stimulating effects on Paramecium were made as the result of the enhancement of the bacterial growth of the clinorotation (Brown et al., 2002). It might be inferred that the reduced proliferation presented in this paper is specific to the condition of the present experiment, where the rotation rate was chosen in close relation to the time scale of reorienting behavior of Paramecium. Hemmersbach-Krause et al. (1993) observed an
1169
increased swimming velocity in a fast rotating clinostat. Another possibility might be raised that the interaction to the chamber walls (e.g. collision to the wall surface) affects differently depending on the rotation rate. Kato et al. (2003) discussed the inhibitory effect of hypergravity in terms of the energetics of the proliferation as well as the motile activities of the cell. They explained that the effect was caused by the decrease in the energy for proliferation in association with the increase in the expenditure of propulsive energy as the result of the enhancement of propulsive thrust due to gravikinetic response to the increased gravity. However, we can not simply explain the inhibitory effect of clinorotation on the proliferation of Paramecium on the basis of the same story as Kato et al. (2003), because swimming velocity was found to be reduced under clinorotation. Ooya et al. (1992) postulated a physiological model of gravitaxis, in which the gravity-dependent membrane potential shift causes changes in pitch angle of helical swimming trajectories as a result of the changes in ciliary motility strongly coupled to the membrane potential. Using electrophysiological data on the ciliary electromotor coupling, computer simulation of the model demonstrated that cells swim preferentially upward along the super-helical trajectories without taking account of any mechanical properties for upward orientation (Mogami and Baba, 1998). If this is the case, Paramecium could change the propulsive activity to orient upwards in response to the forced orientation induced by fluid rotation. This may act as the excess cost for gravity-dependent regulation of propulsion, and may also explain the inhibition of proliferation under clinorotation even with the observed decrease in the translational propulsive velocity. Acknowledgements We thank space project unit of Chiyoda Advanced Solutions Corporation for excellent technical support. This work was carried out as a part of ‘‘Ground-based Research Announcement for Space Utilization’’ promoted by Japan Space Forum. References Ayed, M., Pironneau, O., Planel, H., et al. Theoretical and experimental investigations on the fast rotating clinostat. Microgravity Sci. Technol. 5/2, 98–102, 1992. Brown, R.B., Klaus, D., Todd, P. Effects of space flight, clinorotation, and centrifugation on the substrate utilization efficiency of E. coli. Microgravity Sci. Technol. 13/2, 24–29, 2002. Fok, A.K., Allen, R.D. Axenic Paramecium caudatum. I. Mass culture and structure. J. Protozool. 26, 463–470, 1979. Hemmersbach-Krause, R., Briegleb, W., Ha¨der (1), D.-P., et al. Cellular functions of Paramecium under different gravity conditions, in: Proceeding of the 4th Europ. Symp. of Life Science Research in Space, ESA SP-307, pp. 285–290, 1990. Hemmersbach-Krause, R., Briegleb, W., Vogel, K., et al. Swimming velocity of Paramecium under the condition of weightlessness. Acta Protozool. 32, 229–236, 1993.
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Kato, Y. Cellular responses of Paramecium to the altered gravity. Doctoral Thesis in Ochanomizu Univ. 2004. Kato, Y., Mogami, Y., Baba, S.A. Responses to hypergravity in proliferation of Paramecium tetraurelia. Zool. Sci. 20, 1373–1380, 2003. Kessler, J.O. Individual and collective dynamics of swimming cells. J. Fluid Mech. 173, 191–205, 1986. Kessler, J.O., Hill, N.A., Strittmatter, R., et al. Sedimenting particles and swimming microorganisms in a rotated fluid. Adv. Space Res. 21, 1269–1275, 1998. Machemer, H. Bioelectric control of the ciliary cycle. Lect. Notes Biomath. 89, 169–183, 1990. Machemer, H., Machemer-Roenisch, S., Braeucker, R., et al. Gravikinesis in Paramecium: theory and isolation of physiological response to the natural gravity vector. J. Comp. Physiol. A 168, 1–12, 1991. Mogami, Y., Baba, S.A. Super helix model: a physiological model for gravitaxis of Paramecium. Adv. Space Res. 21, 1291–1300, 1998. Mogami, Y., Komiya, H., Tokunaga, N., et al. Methodology of the cell culture and counting for the analyses on the longevity of cell clone in space. Space Utilization Res. 16, 19–22, 2000. Mogami, Y., Ishii, J., Baba, S.A. Theoretical and experimental dissection of gravity-dependent mechanical orientation in gravitactic microorganisms. Biol. Bull. 201, 26–33, 2001. Ooya, M., Mogami, Y., Izumi-Kurotani, A., et al. Gravity-induced changes in propulsion of paramecium caudatum: a possible role of
gravireception in protozoan behavior. J. Exp. Biol. 163, 153–167, 1992. Planel, H., Tixador, R., Nefedov, Y., et al. Space flight effects on Paramecium tetraurelia flown aboard Salyut 6 in the Cytos I and Cytos M experiments. Adv. Space Res. 1, 95–100, 1981. Planel, H., Tixador, R., Richoilley, G., et al. Effects of space flight on a single-cell organism: Paramecium tetraurelia, in: Asashima, M., Malacinski, G.M. (Eds.), Fundamentals of Space Biology. Japan Sci. Soc. Press/Springer, Tokyo/Berlin, pp. 85–96, 1990. Richoilley, G., Tixador, R., Gasset, G., et al. Preliminary results of the ‘‘Paramecium’’ experiment. Naturwissenschaften 73, 404–406, 1986. Richoilley, G., Planel, H., Gasset, G. Effects of hypergravity on Paramecium tetraurelia. CR Acad. Sci. Paris, Sciences de la vie/Life sciences 316, 585–587, 1993. Roberts, A.M. Geotaxis in motile micro-organisms. J. Exp. Biol. 53, 687–699, 1970. Shiba, K., Ohmuro, J., Yoshihiro, Mogami., et al. Sperm-activating peptide induces asymmetric flagellar bending in sea urchin sperm. Zool. Sci. 22, 293–299, 2005. Soldo, A.T., Godoy, G.A., van Wagtendonk, W.J. Growth of particlebearing and particle-free Paramecium aurelia in axenic culture. J. Protozool. 13, 492–497, 1966. Tixador, R., Richoilley, G., Gasset, G., et al. Effects of gravity and cosmic rays on cell proliferation kinetics in Paramecium tetraurelia. Adv. Space Res. 4, 91–95, 1984.
Cell proliferation of Paramecium tetraurelia on a slow rotating clinostat Satoe Sawai a
a,*
, Yoshihiro Mogami b, Shoji A. Baba
a
Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka 2-1-1, Tokyo 112-8610, Japan b Department of Biology, Ochanomizu University, Otsuka 2-1-1, Tokyo 112-8610, Japan Received 26 September 2006; received in revised form 6 January 2007; accepted 7 February 2007
Abstract Paramecium is known to proliferate faster under microgravity conditions, and slower under hypergravity. Experiments using axenic culture medium have demonstrated that hypergravity affected directly on the proliferation of Paramecium itself. In order to assess the mechanisms underlying the physiological effects of gravity on cell proliferation, Paramecium tetraurelia was grown under clinorotation (2.5 rpm) and the time course of the proliferation was investigated in detail on the basis of the logistic analysis. On the basis of the mechanical properties of Paramecium, this slow rate of the rotation appears to be enough to simulate microgravity in terms of the randomization of the cell orientation with respect to gravity. P. tetraurelia was cultivated in a closed chamber in which cells were confined without air bubbles, reducing the shear forces and turbulences under clinorotation. The chamber is made of quartz and silicone rubber film; the former is for the optically-flat walls for the measurement of cell density by means of a non-invasive laser optical-slice method, and the latter for gas exchange. Because of the small dimension for culture space, Paramecium does not accumulate at the top of the chamber in spite of its known negative gravitactic behavior. We measured the cell density at regular time intervals without breaking the configuration of the chamber, and analyzed the proliferation parameters by fitting the data to a logistic equation. As a result, P. tetraurelia showed reduced proliferation under slow clinorotation. The saturation of the cell density as well as the maximum proliferation rate decreased, although we found no significant changes on the half maximal time for proliferation. We also found that the mean swimming velocity decreased under slow clinorotation. These results were not consistent with those under microgravity and fast rotating clinostat. This may suggest that randomization of the cell orientation performed by slow rotating clinostat has not the same effect on Paramecium as that under microgravity that may affect the proliferation as the result of the reduced cost of propulsion. 2007 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: Paramecium; Clinorotation; Simulated microgravity; Proliferation rate; Swimming velocity
1. Introduction Previous space experiments aboard the Soviet orbital station Salyut 6 (CYTOS experiment) and the Space Shuttle (D1 mission) reported that the proliferation of Paramecium became faster (about 160%) under microgravity in space (Planel et al., 1981; Richoilley et al., 1986). The same authors reported that proliferation became slower (about 70%) under hypergravity (20g) provided by centrifugation (Tixador et al., 1984; Planel et al., 1990; Richoilley et al.,
*
Corresponding author. E-mail address: [email protected] (S. Sawai).
1993). Experiments using axenic culture medium have demonstrated the direct effect of hypergravity on the proliferation of Paramecium itself, other than the indirect effect on the proliferation of nutrient bacteria grown in non-axenic culture (Richoilley et al., 1993; Kato et al., 2003). Mechanisms underlying the gravity dependent changes in proliferation could be explained in terms of the energetics of the proliferation as well as of the motile activities of the cell. Because Paramecium modulate their propulsive thrust depending on the orientation of the cell body with respect to the gravity vector, i.e. increasing propulsive thrust in upward swimming and decreasing it in downward (Machemer et al., 1991; Ooya et al., 1992). This gravityinduced change in propulsion, gravikinesis, is explained
0273-1177/$30 2007 COSPAR. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.asr.2007.02.023
S. Sawai et al. / Advances in Space Research 39 (2007) 1166–1170
on the basis of cellular mechanosensitivity in combination with close coupling between the membrane potential and ciliary locomotors activity (Machemer, 1990). It seems likely that Paramecium consumes much energy when swimming upwards than that when swimming downwards. Mogami et al. (2001) demonstrated that the gravitactic orientation of Paramecium is mechanically biased by the torque mainly due to the fore-aft asymmetry of the cell body (gravitaxis). The upward-orienting torque increases with increase in gravity acceleration, so that the fraction of the upward-orienting cell would decrease under reduced gravity, and increase under hypergravity. This would result in a change in energy expenditure for propulsion under different gravity accelerations. Thrust force would be increased under hypergravity and hence reduced under microgravity. If the energy available for physiological events of the cell is limited, changes in the energy for propulsion may lead to the changes in the energy stock for the proliferation. The larger stock of energy under microgravity as the result of the reduced propulsive work might enhance the proliferation activity and smaller stock would reduce this activity under hypergravity. In order to assess the feasibility of the assumption above, Paramecium tetraurelia was grown under simulated microgravity performed by clinorotation. If it is the case, we may expect the cell proliferation of Paramecium to be enhanced by randomization of the cell orientation by means of clinorotation. 2. Materials and methods P. tetraurelia was cultivated axenically as described previously (Kato et al., 2003) in the medium of Soldo et al. (1966) with the modifications by Fok and Allen (1979). We used a closed chamber for the cell culture under clinorotation in which cells were confined without air bubbles for reducing the shear forces and turbulences under rotation (Fig. 1). The chamber had a culture space with an inner dimension of 3 · 3 · 60 mm, which had an inlet and outlet for the medium. Three walls of the culture space were made of quartz and the rest of silicone rubber film.
a
Moisture space
1167
The quartz walls provided the optically-flat windows for the measurement of cell density by means of a non-invasive laser optical-slice method (Mogami et al., 2000; Kato et al., 2003). Gas exchange for respiration was carried out through the silicone rubber film between the medium and the moist air in the space placed next to the culture space. This prevented the formation of air bubbles in the culture medium caused by the evaporation of water in association with gas exchange. The depth of the culture volume was restricted to 3 mm when the chamber was placed horizontally. In this shallow space, Paramecium showed roughly even vertical distribution rather than the apparent accumulation at the top of the culture volume as observed in a deep water column. The culture chamber was placed on a rotator (RT-5 TAITEC, Tokyo) with the long axis of the chamber kept horizontal and rotated around the long axis (clinorotation) or around the short axis perpendicular to the long axis (control rotation) (Fig. 2). Both the direction and the speed of rotation (2.5 rpm) were kept constant throughout an experiment. For the horizontal rotation, the chamber was placed with the silicone rubber as the side wall of the culture space. Cell density was measured without breaking the configuration of the chamber by optical-slice method (Kato et al., 2003) with removing the chamber from the rotators. The time course of the cell proliferation under clinorotation was analyzed on the basis of the logistic growth equation. According to the three parameters determined by means of the least squares fitting of the equation to the experimental data as described in Kato et al. (2003), the kinetic parameter of proliferation (saturation cell density, maximum pro-
Culture space
Silicone rubber film
b
Moisture space Silicone rubber film Culture space
Fig. 1. Schematic drawings of the closed chamber for the culture of Paramecium under clinorotation. Side view (a) and front view (b) of the chamber showing the culture space and moist air space for gas exchange.
Fig. 2. (a) An overview of the rotator developed for clinorotation. Arrow indicates one of the closed chambers. (b) Schematic drawing of the rotation of the culture space in the clinorotation and the control horizontal rotation.
S. Sawai et al. / Advances in Space Research 39 (2007) 1166–1170
3. Results
ð1Þ
where N is the cell density as a function of time, t, and K, a and l are the parameters characteristic of the individual culture. Eq. (1) is derived from a differential equation of dN =dt ¼ lN ð1 N =KÞ;
ð2Þ
where the growth rate is defined by the product of a proportional constant (l, intrinsic growth rate) and the terms one of which is related to the cell density in culture (N) and another to the density dependent inhibitory effect (1 N/ K); e.g., the reduction in available resources. According to the least squares fitting of parameters in Eq. (1) to the experimental data, the kinetic parameters of proliferation, i.e. saturation (maximal) cell density, maximum proliferation rate and half maximal time (the time for half maximal cell density) were calculated as K, lK/4 and (ln a)/l, respectively. As shown in Fig. 4, clinorotation reduced the saturation cell density and the maximum proliferation rate, although we found no significant changes on half maximal time. Similar reduction of the saturation density and the maximum growth rate was also found under hypergravity condition (Kato et al., 2003). However, reduction by hypergravity was associated with the increase in half maximal time. This is a remarkable difference between the inhibitory effects provided by hypergravity and clinorotation. Effects of clinorotation on Paramecium were also found in propulsive activity. The mean swimming velocity of P. tetraurelia in the logarithmic phase of the proliferation under slow rotating clinostat (2.5 rpm) decreased in average to 85.0 ± 4.7% of the control (data from three duplicated or triplicated measurements containing the measurements on >150 individual cells, P < 0.05). The result was not consistent with the hypergravity experiment: P. tetraurelia has higher swimming activity under hypergravity (Kato, 2004).
0.2
P = 0.60
0.15 0.1 0.05 0
30000
400
P < 0.001
300 200 100 0
25000 15000 10000 5000 0
0
24
48 72 time (hrs)
96
Fig. 3. The time course of the cell proliferation under clinorotation (2.5 rpm, filled circle) as well as control horizontal rotation of the same angular velocity (open circle). Means ± SD of seven duplicated or triplicated measurements are shown. Curves are the result of the least squares fitting of the logistic growth equation.
800 600 400 200 0
P = 0.07
P = 0.05 half maximal time (hr)
20000
maximum proliferation rate (cells/ml hr)
cell density (cells/ml)
As reported briefly (Mogami et al., 2000), Paramecium proliferated in a similar manner in the closed culture chamber to that observed in the open surface culture. Maximal cell density found in the steady state was 2–3 · 104 cell/ml, which is similar to the maximal value in the open surface culture (Kato et al., 2003). This means that the closed culture system used in this study is well-suited to the experiment on the proliferation of Paramecium performed under good physiological conditions. Similar profiles of the proliferation were kept in the culture under horizontal rotation; maximal cell density in the steady state with and without horizontal rotation (2.5 rpm), was 2.83 ± 0.82 · 104 (cells/ml, mean ± SD, n = 6) and 2.30 ± 0.82 · 104 (n = 5), respectively (P = 0.3). Since we did not find any difference in the proliferation under horizontal rotation at various angular velocities (0.2–4.8 rpm), we used the culture under horizontal rotation as the control to the culture under clinorotation. Fig. 3 shows the time course of the cell proliferation under slow rotating clinostat (2.5 rpm) as well as under control horizontal rotation. Cell proliferation was reduced under clinorotation. In order to quantitatively assess the reduction, the time course of the cell proliferation under clinorotation and control horizontal rotation were analyzed on the basis of the logistic growth equation
N ðtÞ ¼ K=f1 þ a expðltÞg;
saturation cell density ( 102 cells/ml)
liferation rate and half maximal time) were calculated. Temperature during culture was regulated at 24 C. Swimming speed of P. tetraurelia was measured from the video recordings under a dark-field illumination performed by the laser optical slice. For this purpose, culture space was illuminated vertically by a slit-laser, and cells swimming in any direction within a vertical illumination slit were used for the measurement. Positions of individual cells were determined by a laboratory-made computer assisted tracking software (Shiba et al., 2005), and the average velocity was calculated from the changes in the distances at defined time intervals. Probability of statistical significance (P) was determined using Student’s t-test.
intrinsic growth rate (/hr)
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400 300 200 100 0
Fig. 4. The kinetic parameters of proliferation of P. tetraurelia cultivated under clinorotation (2.5 rpm, filled bar) as well as control horizontal rotation (open bar). Means ± SD obtained from the least squares fitting of Eq. (1) to the seven sets of experiments.
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The differences found in the profiles of the proliferation as well as the propulsive activity suggest that slow clinorotation caused the inhibitory effect of the proliferation of Paramecium depending on the different mechanisms from those working under hypergravity. 4. Discussion Clinorotation has been known to simulate microgravity in space. However, the clinorotation used in the present paper (slow clinorotation) did not confirm the increased proliferation rate of Paramecium in real microgravity (Planel et al., 1981) and on a fast rotation clinostat (>60 rpm) (Ayed et al., 1992; Hemmersbach-Krause et al., 1990). For the gravitactic behavior of Paramecium, clinorotation may affect both the vertical distribution of the whole population and the upward orientation of individual cells. We used closed culture space with a depth of 3 mm, which is much less than the scale depth of the gravitactic distribution of free-swimming Paramecium (Roberts, 1970). As the results, cells were observed to almost evenly distribute vertically as well as horizontally in the culture space placed without rotation. Therefore, clinorotation might not affect any more to change the distribution profile. According to the theoretical studies on the gravitactic orientation of swimming microorganisms in the rotating fluid field (Kessler, 1986; Kessler et al., 1998), organisms tumble with the fluid when the angular velocity of the fluid rotation is greater than the gravity-dependent orientation rate of the organisms. By the analysis of the sedimentation of immobilized Paramecium, the maximum rate of the orientation was measured with 0.08 rad/s (Mogami et al., 2001), which is much smaller than the angular velocity of clinorotation (2.5 rpm = 0.26 rad/s). It is therefore likely that Paramecium oriented randomly with respect to the gravity vector as the result of the clinorotation at this angular velocity. These indicate that the slow clinorotation used in these studies was effective for randomizing the orientation of the cell with keeping the homogenous vertical distribution in the narrow chamber. Ayed et al. (1992) reported a stimulating effect of clinorotation on the proliferation of Paramecium. The effect depends on rotation rate, and they reported about 25% increase in the cell density at the higher angular velocity (80–90 rpm). We confirmed the inhibitory effect of clinorotation up to 4.8 rpm in the configuration used in this study, although the possibility would not excluded that the rotation at higher angular velocity has stimulatory effects. The other possibility would be considered that the stimulating effects on Paramecium were made as the result of the enhancement of the bacterial growth of the clinorotation (Brown et al., 2002). It might be inferred that the reduced proliferation presented in this paper is specific to the condition of the present experiment, where the rotation rate was chosen in close relation to the time scale of reorienting behavior of Paramecium. Hemmersbach-Krause et al. (1993) observed an
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increased swimming velocity in a fast rotating clinostat. Another possibility might be raised that the interaction to the chamber walls (e.g. collision to the wall surface) affects differently depending on the rotation rate. Kato et al. (2003) discussed the inhibitory effect of hypergravity in terms of the energetics of the proliferation as well as the motile activities of the cell. They explained that the effect was caused by the decrease in the energy for proliferation in association with the increase in the expenditure of propulsive energy as the result of the enhancement of propulsive thrust due to gravikinetic response to the increased gravity. However, we can not simply explain the inhibitory effect of clinorotation on the proliferation of Paramecium on the basis of the same story as Kato et al. (2003), because swimming velocity was found to be reduced under clinorotation. Ooya et al. (1992) postulated a physiological model of gravitaxis, in which the gravity-dependent membrane potential shift causes changes in pitch angle of helical swimming trajectories as a result of the changes in ciliary motility strongly coupled to the membrane potential. Using electrophysiological data on the ciliary electromotor coupling, computer simulation of the model demonstrated that cells swim preferentially upward along the super-helical trajectories without taking account of any mechanical properties for upward orientation (Mogami and Baba, 1998). If this is the case, Paramecium could change the propulsive activity to orient upwards in response to the forced orientation induced by fluid rotation. This may act as the excess cost for gravity-dependent regulation of propulsion, and may also explain the inhibition of proliferation under clinorotation even with the observed decrease in the translational propulsive velocity. Acknowledgements We thank space project unit of Chiyoda Advanced Solutions Corporation for excellent technical support. This work was carried out as a part of ‘‘Ground-based Research Announcement for Space Utilization’’ promoted by Japan Space Forum. References Ayed, M., Pironneau, O., Planel, H., et al. Theoretical and experimental investigations on the fast rotating clinostat. Microgravity Sci. Technol. 5/2, 98–102, 1992. Brown, R.B., Klaus, D., Todd, P. Effects of space flight, clinorotation, and centrifugation on the substrate utilization efficiency of E. coli. Microgravity Sci. Technol. 13/2, 24–29, 2002. Fok, A.K., Allen, R.D. Axenic Paramecium caudatum. I. Mass culture and structure. J. Protozool. 26, 463–470, 1979. Hemmersbach-Krause, R., Briegleb, W., Ha¨der (1), D.-P., et al. Cellular functions of Paramecium under different gravity conditions, in: Proceeding of the 4th Europ. Symp. of Life Science Research in Space, ESA SP-307, pp. 285–290, 1990. Hemmersbach-Krause, R., Briegleb, W., Vogel, K., et al. Swimming velocity of Paramecium under the condition of weightlessness. Acta Protozool. 32, 229–236, 1993.
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