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Gravity-controlled gliding velocity in Loxodes
Europ.]. Protisto!' 28, 238-245 (1992) May 22,1992
European Journal of
PROTISTOLOGY
Gravity-controlled Gliding Velocity in Loxodes .Richard Braucker, Sigrun Machemer-R6hnisch and Hans Machemer Arbeitsgruppe Zellulare Erregungsphysiologie, Fakultat fOr Biologie, Ruhr-Universitat, Bochum, FRG
Akira Murakami Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo, Japan
SUMMARY The locomotion of Loxodes as controlled by the natural gravity vector was investigated employing a mass-cell approach. Samples of cells were incubated for 4 hours in a 1.6 mm deep well (41 X 85 mm) filled with defined experimental solution. Their gliding locomotion on surfaces inclined between 00 and 90° was recorded by videocamera. Steady gliding rates (median of total: 206 um/s; n = 12711) were evaluated by calculating the vertical components from observed tracks. At a given inclination the rates of downward and upward gliding were similar. The sedimentation rate of freely suspended nickel-immobilized specimens (S = 49 urn/s), and vertical rates of displacement at 6 differently inclined planes with cells in "sitting" posture as well as "hanging" (upside down) were used to determine the gravity-dependent component (L1 = -49 um/s) during gliding motion. Numerical equality ofthe gravity force to produce Sand of the counterforce to produce L1 follows from the observed constancy of gliding rates (V) and identical medians of vertical displacements during gliding along vertically oriented surfaces (169 urn/s). It is suggested that neutralization of sedimentation might be a favourable condition for migration and accumulation of Loxodes along vertical O 2 gradients in its freshwater habitat.
Introduction
Gravitaxis is common to unicellular organisms such as prokaryotes, protists, fungi and spermatozoa, suggesting the existence of cellular systems for gravitransduction. Extensive research on the responses to gravity in large protozoans such as Paramecium has led to the question whether pure physical principles and/or mechanisms involving sensory perception are candidates for causing migration of cells with respect to the gravity vector. Conclusive evidence of gravisensation and isolation of the presumed physiological component were missing so far (reviews: [1, 12, 20]). Recently, a gravitaxis has been described in the limnic ciliate Loxodes, which is intricately linked to the sensation of chemical and photic stimuli. According to Fenchel and Finlay [4-9], Loxodes aggregates in environments of low O 2 tension, employing 0932-4739/92/0028-023 8$3.50/0
negative and positive gravitaxis, and illumination enhances the avoidance of OJ. The existence of Muller vesicles, that is, statocyst-like organelles ("statocystoids") in the anterior cortex of Loxodes was seen as supportive evidence of sensation and response to the gravity vector in this cell. In the present study, we have investigated the velocity of Loxodes during gliding motion. This type of locomotion prevails over swimming at low O 2 tension, takes a geometrically more regular course, and exceeds swimming in velocity [4]. Gliding is generated by cilia which predominate on the flattened right body side; this side of the cell faces solid substrates during locomotion in the sediments. We attempt to exclude orientational components from analysis of the behaviour, and to control circumstantial conditions which might contribute to stimulation. Our approach is based on established principles of ciliate © 1992 by Gustav Fischer Verlag, Stuttgart
Gra vity Response in Loxodes . 239
sensorimotor coupling [ 16-18] according to which a mechanical input can alte r the membrane potential and thereby the direction and rate of ciliary activity, which modul ates behaviour. In a previous study of Paramecium, we have isolated a kinetic respon se which antagonizes sedimentation and was therefore called a "negative gravikinesis" [22] . Here, we show that a similar biased response exists in Loxodes. Contrary to Paramecium, populations of gliding Loxodes appear to fully compensate the effect of sedimentation.
temp erature inside the chamber was between 22° and 23 °C (N iCr-Ni microprobe; GTH 1200, G reisne r). Locomotion of Lox odes was recor ded on videotap e with the cha mber or iented at 0 the angles of 0°, 15 , 30°, 45 0 , 60 0 , 75 0 , 90 0 for periods of 2 minutes each, and once again after a slow 180° reorientation of th e cha mber. 90 s were allow ed to reset the inclinat ion of the cha mber by 15°. Each 2-min reco rd of cell gliding was used to generate 5 separate repre sent arion s of 5-s tracks from the video tap es by computer program . From each of these superimposed video fields, typic ally 4 to 10 tracks qu alified for quanrirativ e evaluatio n. Selection of the video fields was organized so as to avoid more than one representation o f the sa me specimen. The numbers of readings given in the Figu res and Tables are therefore lar gely coincident with the number of ind ividual cells.
Material and Methods Culture. A wild-type line of Lox odes striatus was kindly supplied by B. J. Finlay (Freshwater Biological Association, Amb leside, Cumbria), and rear ed in soil solutio n (Erdschreiber) at pH 6.8 which was depleted of O 2 by gassing with N z and CO 2 , A fresh culture was kept at 12 °C for 4 8 hours and then tran sferr ed to 22 °C for furth er culturing in the dark. Ten da ys old cultures of Euglena gracilis we re used for feeding. The Loxodes cultu res were read y for experiments two weeks after inocul ation, i.e., in their stationary pha se.
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Ex perimental Solutio n. A so lut ion of 1 mM CaCh + 0.5 mM KCI + 0. 1 mM MgS0 4 + 0.41 mM Na zHP0 4 + 0.18 mM NaH 2P04 at pH 7.0 was used for th e experiments . Cells were concent rated by circular swinging of the culture dishes whi ch leads to accumulation in th e cent er of the dish. Samples of cells were transferred to the experiment al chamber using a syringe . Exp erimental Chamber. The cha mber (Fig. 1A) corresponds to th at used pre viou sly for Paramecium (perspex; 65 x 110 x 15 mm o uter dimensions; fluid space of 41 x 85 x 1.6 mm) [22].
B
Cell Immobilization and Sedim entation. A sample of cells in experimental solution was exposed for 10 min to 1 mM NiCh dissolved in the same solution. and then transferred to the chamber; 20 more minutes wer eallowed for complete imm obilization of the cells in th e chamber. Measur ements of sedimentation in the vertica l chamber started 30 min after beginni ng of the immobili zation procedure. Recording. Gliding locomotion on the chamber bottom or cover surface was record ed by a CC D-videoca mera (Panasonic F 10, 25 Hz framing rate ) connected to a macro-Jens. Th e resultin g magnification was 26 x . A ring of green light-emitting diod es (565 nm ) attached to the rear of the chamber (Fig. 1B) provided a n even dark -field illumin ati on o f the central area of the chamb er enclosing the cells (Fig. 1A). The ca mera and chamber were fixed to a platform, whi ch was hinged so as to allow a rota tion at 15° intervals betw een the hor izontal (90° inclination ) and vert ical chamber po sition (0°; Fig. 1 B). Cells gliding on th e upp er chamber sur face (right side of cell up) or on the lower chamber sur face (right side of cell down ) were identified by appro priate focu sing of the lens (Fig. 1C). Parameters such as experiment identification, date, time, and ori entation of the cha mber were superimposed o n the video field. Exp erimental Protocol. A sample of 150-200 cells was tr ansferr ed to the chamber with a minimum of agitation. In parti cula r, care was taken to avoid mechani cal disturbances, such as vibrations, during equilibrati on a nd the experi ment , The
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c Fig. 1. A. Cha mber showing central space for recording of cells in experimental solution (26 x 41 mm ) a nd the lateral areas filled with aga r (1.5% in expo soluti on; dashed a rea ). B. Hinged mount of video reco rding unit (VC) with darkfield illumin ation by a ring of light-emitting diodes (I) behind th e cha mber (C). The unit ca n be rot ated to set the chamber at an y angle from 0 (= verti cal) to 90°C. Definition of inclination angle (8) and identificat ion o f Loxodes inside the chamber with its right side attached either to the low er (L) or upper chamber surface (U). These attachments a re identified by focusing of the macr o-lens on the distal and pro xim al sur face of the chamber, respectively. 0
240 . R. Briiucker, S. Machemcr-Rohnisch, H. Machemer and A. Murakami
Criteria for the Assessment of Steady Gliding Movement. At low oxygen tension, Loxodes commonly glides along a circular track of large radius (Fig. 2). Gliding is occasionally interrupted by spontaneous reversals. The distance between the beginning and the end of a 5-s track ranged between 0.5 and 1.3 mm. Our aim was to evaluate data from undamaged cells showing even gliding motion. We therefore used the start, middle and end points of tracks of little curvature, excluding reversals, for criteria of suitability for evaluation (Fig. 2B-E; see legend for details of selection).
or downward gliding were calculated using the cosine of the angle subtended between the vertical and the chord of a track (see equ. 4 and Fig. 6).
Evaluation of Data. The positions of, and distance between, start and end of a track (= length of cord) were identified. Then, the suitability for further evaluation was tested by means of image analysis (Fig. 2). The rate and orientation of locomotion were determined. From these data, the vertical components of upward
General Behaviour. After 4 hours of incubation in the chamber (experimental space 1.6 ml; horizontal orientation), Loxodes glides on the chamber surfaces at velocities ranging between 150 and 250 um/s. A minor proportion of the cells swims under these conditions. Mechanical shock may induce free swimming along irregular helical courses and/or contraction suggesting mechanosensitivity of the cell. Cells which have been equilibrated for at least one hour and isolated from uncontrolled stimuli such as vibration, heat or strong incident light, continue to show reversals or jerks at irregular intervals. This behaviour resembles spontaneous excitation and ciliary reversal in other ciliates [19, 21, 25]. Observation of cells in the undisturbed experimental solution or in culture fluid, reveals no signs of behavioural responses to gravity comparable to the negative gravitaxis in Paramecium. Direct inspection of tracks recorded with the chamber held in the horizontal and vertical positions do not suggest the existence of directional differences in gliding velocity (Fig. 3).
c
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Fig. 2. Criteria for isolation of the steady locomotion rate in Loxodes. Typically, cells glide along straight or slighly curved tracks at constant speed (A). Positions of cells at 3 times separated by the same intervals were determined. For evaluations, the following types of traces were eliminated: B. Traces of large curvature (d ~ w). C. Large changes in gliding velocity. D. Very low rate or unusual form of locomotion suggesting damaged cell. E. Traces indicating that a spontaneous reversal occurred during time of recording.
Statistics. We have chosen nonparametric statistics (calculation of median values; 95% confidence range) because gaussian distribution of data is not assured (see [22]).
Results
Gliding Rates. Quantitative evaluation shows that the gliding rates were virtually the same at all inclinations (Table 1). A small difference exists between the rates on the lower surface (205 um/s) and on the upper surface (231 um/s), which also shows up in the orientational distributions of horizontal rates of locomotion (compare Fig. 4A with B). In Fig. 4A, the horizontal gliding rates of those cells which head toward the agar sides of the chamber (Fig. 1A) are slighly reduced as compared to those moving normal to that axis. It is possible, therefore,
A
A
B
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Fig. 3. Samples of traces of Loxodes with the chamber oriented horizontally (A) and vertically (B). An arrow indicates the direction of gliding from the starting position. In the vertical orientation, cells moved predominantly upward or downward. The traces marked by (',.) have been eliminated from the evaluations according to the criteria shown in Fig. 2.
Grav ity Response in Loxodes . 24 1 Table 1. Median gliding velocities (V, um/s) of Loxodes along planes of different inclinatio n (8; see Fig. 6). A. Cells with their ciliated right side down . B. Cells with their right side up . C. Universal median ra te including all orientation s. Th e median s of uncorrected rat es of all directions on one plane are given together with the 95 % con fidence range (n = num ber of independ ent readings). It is seen that the gliding rates were virt ually independent of the inclination angle suggesting full neut ralizati on of sediment ation
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Fig. 4. Pola rograms of cellular gliding rate with the experi mental cha mber oriented hor izontally. A. Cells (n = 1776) with their right body side down, that is, "sitt ing" on the cha mber surface . B. "H anging" cells (n = 80 1) with their right body side up . For compensation of possibl e effects of the environ ment on gliding rates (such as aga r blocks; see Fig. 1A), median values of the rat es of all lSO-sector s have been corrected using the universal median of rates in all directions (V = 206 um/s; n = 1271 1) for reference (transition Fig. 4 ~ Fig. 5). Gliding rat es in Tab le 2 are also multiplied with correspond ing correction factors to offset any non-gravitati onal bias.
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Fig. 5. Velocity distrib ution s of Lox odes (V-values, Fig. 6) gliding at different inclina tions between 90° (horizontal) and 0° (vert ical) with the right body side down (A) and up (B). Th e top of the polaro grams correspo nds to "up" (except for e = 90°). Medians of the gliding rat es in all l .S'-secrors and at all inclinations are corrected for neutrality toward a non-gravitation al bias (see Fig. 4). Not e that up and down gliding rat es were virt ually unaffected by the inclination angle, or whether the cell was "s itti ng" (A) or " hanging" (B). Figures below the polaro grams are the numb er of velocity readings (n). Inset at lower right gives the velocity calibration of the pola rograms.
242 . R. Braucker, S. Macherner-Rohnisch, H. Machemer and A. Murakami
that the agar blocks established, during equilibration of the cells, minor gradients in aeration (02?, CO 2?) in the recording field. This should, in fact, be seen in horizontal cells, which are assumed to be minimally affected by gravity [22]. In order to offset in the graphical display (Fig. 5) an unknown non-gravitational bias on locomotion, and to allow a comparison of velocity distributions between cells in "sitting" (Fig. SA) and in "hanging" inclination (Fig. 5B), we have normalized the horizontal gliding rates using the universal median of velocities in all directions (206 um/s; n = 12711) for horizontal reference. Correspondingly, correction factors have been employed in Fig. 5 for each of the 24 orientational sectors at 6 angles of inclination (75 to 0 Figure 5 illustrates the overall permanence of the velocity irrespective of the angle of inclination, upward or downward locomotion, and whether the substrate for gliding was below (Loxodes "sitting") or above the cell ("hanging"). With the chamber at very steep inclination (150, 0 0), the rates of upward and downward gliding tended to exceed those occurring perpendicular to this axis. It is possible that errors in identification of gliding cells from those which occasionally separate from the chamber surface and start free swimming have contributed to this minor departure from radial symmetry of the 15 and 0 polarograms (Fig. SA). In depicting essentially the same rates upward and downward, Fig. 5 suggests that Loxodes can compensate effects of gravity on gliding locomotion. Table 1 supplements this conclusion in listing the median velocities at different inclinations of the plane of gliding. 0
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Sedimentation. A simple explanation of the behaviour shown in Fig. 5 might be a neutral buoyancy of Loxodes in the experimental solution. Tests of sedimentation using nickel-immobilized cells established that this assumption does not apply. Loxodes sedimented at a rate of 49 urn/s (Table 2). This value represents the median (+ 7, - 5 um/s; 95% confidence range; n = 312). A similar median value of 47 [!m/s resulted from sedimentations of Loxodes in a different type of chamber (n = 963; unpublished observations). Because sedimentation of freely suspended immobilized cells corresponds to as much as 24% of the gliding rate (median: 206 um/s; Table 1), gravitational pull may affect gliding motion of Loxodes, tending to augment downward displacement and to diminish upward displacement. Determination ofa Gravity-Induced Kinesis. In order to compensate for effects of the gravity force during gliding locomotion as documented in Fig. 5, Loxodes is likely to generate forces equal to those which produce sedimentation, but act in the opposite direction. Irrespective of the actual size of sedimentation during gliding, which cannot be measured, these forces may be represented, under conditions of low Reynolds numbers, in terms of a negative gravikinesis [22].
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Table 2. Vertical components (0, U) of gliding rates of Loxodes, sedimentation (5) and the gravikinetic response (L1) as isolated from gliding on surfaces inclined by the angle e (equation 3). Cells with their ciliated right side down (A) or up (B). Dimensions: um/s; number of readings in brackets. Medians of the downward (0') and upward components (U') along the fall-line of a surface are calculated from individual gliding rates (V;), corrected according to Fig. 5, and multiplied with the cosine of the declination angle a (Fig. Ga). 0 and U are medians of vertical down and up components as calculated after inclusion of the inclination angle e (Fig. 6B). Note that in the 11 different groups, as defined by inclination angle and orientation of the cell body, the resulting value of L1 approximates the sedimentation rate
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Gra vity Respon se in Loxo des . 243
where D and U are the vertical comp onent s of downward and upward gliding velocities, P is the vertical component of the rate of propulsion free of the grav ita tional bias, S is the sediment ation rate , L10 and L1u repre sent the gra vityindu ced downward and upw ard respon ses along the vert ical. Equ ation 3 results from summing of equations 1 and 2 introducing L1 as th e mean of L10 and L1u. For any glid ing track in space, D and U are cosine functions of the indi vidu al declinati on angle (a ) from the fall-line of the chamb er (D', U' ; Fig. 6A) and the inclination (0) of the cha mber (Fig. 6 B):
t = Vi cosa· case , U i = Vi cosa· case,
u
v
A
D
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wh ere Vi represents the veloc ity of individual gliding . Table 2 shows the median valu es of D' and U', and of D and D, as calculated for 11 specified planes. The velocitie s along the fall-lines, D' and U', are similar for all inclinations, whereas corresponding values of pa irs of D and U (= the median vert ical rat es) decrease with rising inclination (0°: 169 um/s; 75°: near 39 urn/s). Using the sedimentati on rate of 49 ~lm/s and equa tion 3, negat ive med ian values of L1 (i.e., acting inverse to the direction of sedimenta tion) are calcul at ed for each specified plane. These rat es cent er about the value of -49 um/s. Appli cation of equation 3 for the data show n in Fig. 5 suggests an active neutralization of gravity-indu ced forces of sedimentati on of gliding cells. With L1 being defined as the arithmetic mean of L10 and L1 u, how can these subcompo nents be ident ified? Fig. 7 illustrates the notion that, in order to correspo nd to the observed similar downward and upward gliding velocities in any direction (Fig. 5 ), L10 and L1 u must neutralize sedimenta tion, so that L1 =: L1 0 = L1 u. For determinations of L10 and L1u, we assume that the median of gliding in all
B Fig. 7. Scheme to expl ain how ra tes o f upwa rd or down ward gliding (V) in Loxodes can be regulat ed so as to attain a constant value. The grav ity-induced fraction s ( ~1I, ~D ) of th e vertica l compo nents of active propulsion (U, D) a re equal and inverse to S. Thi s ap plies even if the value of S dur ing gliding might differ fro m th at determ ined in the free fluid. A. Upwa rd gliding in arbitra ry dir ection. B. Down ward gliding in arbitrary direction .
direction s (206 + 2, -3 um/s; n = 12711 , Table 1) is a reasonably good app roximat ion of cellular propul sion (P) as unaffected by grav ity. Multiplicati on of P with the cosine of inclination and declinat ion (cos a . costl; equ, 4) then lead s to D and U, respectively, so that, according to
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Fig. 6. Determ inati on of vert ical upward (U) a nd downward (D) gliding rates. A. For gliding on an inclined plan e, th e individual rate (Vi) is tran sformed to U; or D; (= pa rallel to fall-lin e) by mult iplicati o n with th e cosine of the declinat ion angle (a) . B. Side view of (A). The term of U; transforms to U, an d D; to Di , by multiplicat ion with the cosine of the inclination angle (8).
244 . R. Braucker, S. Machemer-Rohnisch, H. Macherner and A. Murakami
equations 1 and 2, Llo and Llu each cancel sedimentation. Discussion Our data show that Loxodes is able to neutralize fully gravitational effects during gliding locomotion. This property of the sensorimotor organization is beneficial to Loxodes, which migrates toward levels of low oxygen tension for food intake and multiplication [8]. Fenchel and Finlay describe locomotion in Loxodes as a random walk, which is modulated by oxygen tension and light intensity [6-9]. To us, it appears to be quite obvious that vertical migrations biased by O 2 and light occur on the precondition that Loxodes manages to offset the disturbance of locomotion due to passive sedimentation. The existence of an inverse strategy has been suggested by Fenchel and Finlay: modulation of gravitaxis of Loxodes (1) by the surrounding O 2 tension (high 02: positive gravitaxis; low 02: negative gravitaxis; Oj-optimum: no gravitaxis; [4,9]), and (2) by light (which shifts the preferred O 2 tension toward even lower concentration levels; [6,8]). More detailed knowledge on the differential effects of gravity, oxygen, and light on gliding, swimming and reversals, respectively, is desirable for full elucidation of the roles of these different stimulus modalities in guiding Loxodes cells to aggregate in their preferred environment.
Terminology. In a previous paper dealing with gravity as affecting locomotion in Paramecium [22], we have called the gravity-induced modulation of active propulsion rates a kinesis, and we tend to apply the same term to the gravity-responses described in this paper. Conventionally, only nondirectional stimuli are thought to induce a kinesis [3,28], whereas we postulate that morphological and physiological organizations of the cell and properties of the stimulus determine a kinetic response in a manner which is difficult to anticipate a priori. Our data suggest that the gravity vector modulates gliding velocity so as to antagonize sedimentation. In this respect, this non-orienting velocity response would resemble a taxis, and we propose to call it a negative gravikinesis. Gravikinesis may be an intermediate between conventional kinesis (nondirectional stimulus, non-orienting response) and taxis (directional stimulus, orienting response). The Problem of Sedimentation during Gliding Motion. In free swimming cells, such as Paramecium, sedimentation directly subtracts from upward swimming, and adds to downward swimming. Locomotion of a cell which glides along a solid substrate may differ from swimming in the unbounded fluid. According to the hydrodynamics of motion at low Reynolds numbers, a self-propelled small body is likely to increase its velocity in the vicinity of a wall due to the unstirred layer of water near a solid boundary (= wall effect); the velocity of an externally driven body of the same size, on the other hand, may decrease near a wall [27,30]. Preponderance in Loxodes of the gliding rate (206 um/sl.over the swimming rate (70-180 um/s; [4])
agrees with hydrodynamic predictions. It also follows that our value of passive sedimentation (S = 49 um/s), which was determined with the immobilized cells freely suspended, might not fully apply to gliding cells, where some reduction of sedimentation would be expected. It is easily seen from Fig. 7 that, with our observations of constant gliding velocities (Fig. 5), a reduced sedimentation implies a decrease of the gravikinetic response by the same amount. In the extreme and unlikely case that S = 0, both Llo and Llu would be reduced to zero to comply with the observations. The rate of sedimentation during gliding remains unknown for principal reasons. Irrespective of the sedimentation component in a gliding cell, the effective gravitational force (downward) needs to be cancelled by an active counterforce (upward). With the density of Loxodes cells ranging between 1.01 and 1.02 glml (preliminary, unpublished data), the pressure of the cytoplasm on the lower membrane of a vertically oriented cell is in the order of 0.03 Pa (or 0.3 dyne/cm-). This gravitational force is neutralized by an extra kinetic effort of the cilia, which may be expressed in terms of an increment (or decrement) in rate of locomotion, which the same ciliary work is able to generate. The existence of a gravikinetic response in Loxodes, albeit invisible in gliding under natural gravity, is further supported by observations during centrifugation showing that, with rising gravity and increasing rates of sedimentation, the upward gliding rates were unchanged, whereas the downward gliding rates continuously increased (unpublished data).
Possible Electrophysiological Implications. Until now, Loxodes has not been investigated electrophysiologically. The values of LlD and Llu are so far unrelated to sites and functions of sensory transduction. In Paramecium, Stylonychia, Stentor and Didinium, the anterior soma membrane bears depolarizing mechanoreceptor channels [2, 11, 24, 29]. Paramecium and Stylonychia, in addition, show hyperpolarizing mechanoreceptor responses following stimulation of the posterior soma. Small depolarizations from the resting potential depress forward locomotion, hyperpolarizations augment swimming in Paramecium [21,23]. With determinations of vertical and horizontal swimming rates, and of sedimentation, at hand in Paramecium, it has been argued that the gravitational load of the cytoplasm on the lower (= anterior) membrane depolarizes downward-swimming cells, and hyperpolarizes the lower (= posterior) membrane in cells swimming upward [22]. Our calculation of the value and sign of Llo and Llu, agrees with the electrophysiologically sound hypothesis that Loxodes generates a steady positive-going gravireceptor potential during downward gliding, and a negative-going gravireceptor potential during upward gliding. Loxodes bears statocystoids (" Muller vesicles") near the anteriorventral cell end, and these organelles have been implicated in gravisensation theories [4,5]. Assuming, in accordance with the statocyst hypothesis of gravisensation [13-15], that gravity-induced deformation of a mechanosensory substrate constitutes the adequate stimulus, the statocystoids of Loxodes, which enclose a statolith of
Gravity Response in Loxodes . 245 BaS04 [10,26], may have a crucial function in gravisensory transduction [5]. More conventional transduction principles like stretch deformation of the anterior and posterior soma membrane, analogous to mechanotransduction in Paramecium, may be envisioned as well. Physiological probing into Loxodes including the roles of the Muller vesicles, and detailed behavioural studies are desirable to help unveiling the mechanism of gravitransduction in this cell. Acknowledgements This work was supported by the Bundesminister fur Forschung und Technologie, Federal Republic of Germany, grant QV 8857 (to HM), and the Special Fund for Promoting Science and Technology, STA, Japan (to AM). We thank P. F. M. Teunis for critical discussions and G. Krumbach, U. Schilken and P. Ullrich for devoted technical assistance.
References 1 Bean B. (1984): Microbial geotaxis. In: Colombetti G. and Lenci F. (eds.): Membranes and sensory transduction, pp. 163-198. Plenum, New York, London. 2 De PeyerJ. E. and Machemer H. (1978): Hyperpolarizing and depolarizing mechanoreceptor potentials in Stylonychia. J. Compo Physiol., 127,255-266. 3 Diehn B., Feinleib M., Haupt W., Hildebrand E., Lenci F. and Nultsch W. (1977): Terminology of behavioral responses of motile microorganisms. Photochem. Photobiol., 26, 559-560. 4 Fenchel T. and Finlay B. J. (1984): Geotaxis in the protozoon Loxodes. J. Exp. Bio!., 110, 17-33. 5 Fenchel T. and Finlay B. J. (1986a): The structure and function of Miiller vesicles in loxodid ciliates. J. Protozoo!., 33,69-76. 6 Fenchel T. and Finlay B. J. (1986b): Photobehavior of the ciliated protozoon Loxodes: taxic, transient, and kinetic responses in the presence and absence of oxygen. J. Protozoo!., 33, 139-145. 7 Finlay B. J. and Fenchel T. (1986a): Photosensitivity in the ciliated protozoon Loxodes: pigment granules, absorption and action spectra, blue light perception, and ecological significance. J. Protozool., 33, 534-542. 8 Finlay B. J. and Fenchel T. (1986b): Physiological ecology of the ciliated protozoon Loxodes. Rep. Freshwat. Bio!.Ass., 54, 73-96. 9 Finlay B. J., Fenchel T. and Gardener S. (1986): Oxygen perception and O 2 toxicity in the freshwater ciliated protozoon Loxodes. J. Protozoo!., 33, 157-165. 10 Finlay B. J., Hetherington N. B. and Davison W. (1983): Active biological participation in lacustrine barium chemistry. Geochim. Cosmochim. Acta, 47, 1325-1329. 11 Hara R. and Asai H. (1980): Electrophysiological responses of Dtdinium nasutum to Paramecium capture and mechanical stimulation. Nature, 283, 869-870.
12 Haupt W. (1962): Geotaxis. In: Ruhland W. (cd.): Encyclopedia of plant physiology, vo!. XVII. Physiology of movements, pp. 390-395. Springer Verlag, Berlin. 13 Koehler O. (1922): Dber die Geotaxis von Paramecium. Arch. Protistenk., 45, 1-94. 14 Loeb J. (1897): Zur Theorie der physiologischen Licht- und Schwerkraftwirkungen. Pflugers Arch. Ges. Physio!., 66, 439-466. 15 Lyon E. P. (1905): On the theory of geotropism in Paramecium. Am. J. Physiol., 14,421-432. 16 Machemer H. (1986): Electromotor coupling in cilia. In: Luttgau H. C. (ed.): Membrane control of cellular activity. Fortschr. Zoo!., 33, 205-250. 17 Machemer H. (1988): Motor control of cilia. In: Gortz H. D. (ed.): Paramecium, pp. 216-235. Springer Verlag, Berlin. 18 Machemer H. and Deitmer J. W. (1985): Mechanoreception in ciliates. In: Autrum H., Ottoson D., Perl E. R., Schmidt R. F., Shimazu H. and Willis W. D. (eds.): Progress in sensory physiology, vo!. 5, pp. 81-118. Springer Verlag, Berlin. 19 Machemer H. and Deitmer J. W. (1987): From structure to behaviour: Stylonychia as a model system for cellular physiology. In: Corliss J. O. and Patterson D. J. (cds.): Progress in protistology, vo!. 2, pp. 213-330. Biopress, Bristo!' 20 Machemer H. and De Peyer J. E. (1977): Swimming sensory cells: electrical membrane parameters, receptor properties and motor control in ciliated protozoa. Verh. Dtsch. Zoo!' Ges., 1977,86-110. 21 Machemer H. and Sugino K. (1989): Electrophysiological control of ciliary beating: a basis of motile behaviour in ciliated protozoa. Compo Biochem. Physiol., 94A, 365-374. 22 Machemer H., Machemer-Rohnisch S., Braucker R. and Takahashi K. (1991): Gravikinesis in Paramecium: theory and isolation of a physiological response to the natural gravity vector. J. Compo Physiol, A, 168, 1-12. 23 Machemer-Rohnisch S. and Machemer H. (1989): A Ca paradox: electric and behavioural responses of Paramecium to changes in cation concentration of the medium. Europ. J. Protisto!', 25, 45-59. 24 Naitoh Y. and Eckert R. (1969): Ionic mechanisms controlling behavioral responses in Paramecium to mechanical stimulation. Science, 164,963-965. 25 Pape H. C. and Machemer H. (1986): Electrical properties and membrane currents in the ciliate Didinium. J. Compo Physiol, A, 158, 111-124. 26 Rieder N., Ott H. A., Pfundstein P. and Schoch R. (1982): X-ray microanalysis of the mineral contents of some protozoa. J. Protozoo!., 29,15-18. 27 Roberts A. M. (1981): Hydrodynamics of protozoan swimming. In: Levandowsky M. and Hutner S. H. (eds.): Biochemistry and physiology of protozoa, vo!. 4, pp. 5-66. Academic Press, New York. 28 Schone H. (1980): Orientierung im Raum (Hall W., ed.), pp. 1-377. Wissenschaftliche Verlagsgesellschaft, Stuttgart. 29 Wood D. C. (1982): Membrane perrneabilities determining resting, action and mechanoreceptor potentials in Stentor coeruleus. J. Compo Physiol., 146, 537-550. 30 Wu Y. T. (1977): Hydrodynamics of swimming at low Reynolds numbers. In: Nachtigall W. (ed.): Physiology of movement - biomechanics. Fortschr. Zoo!', 24, 149-169.
Key words: Mechanosensation - Gravitaxis - Loxodes Richard Brauker, Arbeitsgruppe Zellulare Erregungsphysiologie, Fakulrat fiir Biologie, Ruhr-Universitat, D-4630 Bochum 1, FRG
European Journal of
PROTISTOLOGY
Gravity-controlled Gliding Velocity in Loxodes .Richard Braucker, Sigrun Machemer-R6hnisch and Hans Machemer Arbeitsgruppe Zellulare Erregungsphysiologie, Fakultat fOr Biologie, Ruhr-Universitat, Bochum, FRG
Akira Murakami Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo, Japan
SUMMARY The locomotion of Loxodes as controlled by the natural gravity vector was investigated employing a mass-cell approach. Samples of cells were incubated for 4 hours in a 1.6 mm deep well (41 X 85 mm) filled with defined experimental solution. Their gliding locomotion on surfaces inclined between 00 and 90° was recorded by videocamera. Steady gliding rates (median of total: 206 um/s; n = 12711) were evaluated by calculating the vertical components from observed tracks. At a given inclination the rates of downward and upward gliding were similar. The sedimentation rate of freely suspended nickel-immobilized specimens (S = 49 urn/s), and vertical rates of displacement at 6 differently inclined planes with cells in "sitting" posture as well as "hanging" (upside down) were used to determine the gravity-dependent component (L1 = -49 um/s) during gliding motion. Numerical equality ofthe gravity force to produce Sand of the counterforce to produce L1 follows from the observed constancy of gliding rates (V) and identical medians of vertical displacements during gliding along vertically oriented surfaces (169 urn/s). It is suggested that neutralization of sedimentation might be a favourable condition for migration and accumulation of Loxodes along vertical O 2 gradients in its freshwater habitat.
Introduction
Gravitaxis is common to unicellular organisms such as prokaryotes, protists, fungi and spermatozoa, suggesting the existence of cellular systems for gravitransduction. Extensive research on the responses to gravity in large protozoans such as Paramecium has led to the question whether pure physical principles and/or mechanisms involving sensory perception are candidates for causing migration of cells with respect to the gravity vector. Conclusive evidence of gravisensation and isolation of the presumed physiological component were missing so far (reviews: [1, 12, 20]). Recently, a gravitaxis has been described in the limnic ciliate Loxodes, which is intricately linked to the sensation of chemical and photic stimuli. According to Fenchel and Finlay [4-9], Loxodes aggregates in environments of low O 2 tension, employing 0932-4739/92/0028-023 8$3.50/0
negative and positive gravitaxis, and illumination enhances the avoidance of OJ. The existence of Muller vesicles, that is, statocyst-like organelles ("statocystoids") in the anterior cortex of Loxodes was seen as supportive evidence of sensation and response to the gravity vector in this cell. In the present study, we have investigated the velocity of Loxodes during gliding motion. This type of locomotion prevails over swimming at low O 2 tension, takes a geometrically more regular course, and exceeds swimming in velocity [4]. Gliding is generated by cilia which predominate on the flattened right body side; this side of the cell faces solid substrates during locomotion in the sediments. We attempt to exclude orientational components from analysis of the behaviour, and to control circumstantial conditions which might contribute to stimulation. Our approach is based on established principles of ciliate © 1992 by Gustav Fischer Verlag, Stuttgart
Gra vity Response in Loxodes . 239
sensorimotor coupling [ 16-18] according to which a mechanical input can alte r the membrane potential and thereby the direction and rate of ciliary activity, which modul ates behaviour. In a previous study of Paramecium, we have isolated a kinetic respon se which antagonizes sedimentation and was therefore called a "negative gravikinesis" [22] . Here, we show that a similar biased response exists in Loxodes. Contrary to Paramecium, populations of gliding Loxodes appear to fully compensate the effect of sedimentation.
temp erature inside the chamber was between 22° and 23 °C (N iCr-Ni microprobe; GTH 1200, G reisne r). Locomotion of Lox odes was recor ded on videotap e with the cha mber or iented at 0 the angles of 0°, 15 , 30°, 45 0 , 60 0 , 75 0 , 90 0 for periods of 2 minutes each, and once again after a slow 180° reorientation of th e cha mber. 90 s were allow ed to reset the inclinat ion of the cha mber by 15°. Each 2-min reco rd of cell gliding was used to generate 5 separate repre sent arion s of 5-s tracks from the video tap es by computer program . From each of these superimposed video fields, typic ally 4 to 10 tracks qu alified for quanrirativ e evaluatio n. Selection of the video fields was organized so as to avoid more than one representation o f the sa me specimen. The numbers of readings given in the Figu res and Tables are therefore lar gely coincident with the number of ind ividual cells.
Material and Methods Culture. A wild-type line of Lox odes striatus was kindly supplied by B. J. Finlay (Freshwater Biological Association, Amb leside, Cumbria), and rear ed in soil solutio n (Erdschreiber) at pH 6.8 which was depleted of O 2 by gassing with N z and CO 2 , A fresh culture was kept at 12 °C for 4 8 hours and then tran sferr ed to 22 °C for furth er culturing in the dark. Ten da ys old cultures of Euglena gracilis we re used for feeding. The Loxodes cultu res were read y for experiments two weeks after inocul ation, i.e., in their stationary pha se.
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Ex perimental Solutio n. A so lut ion of 1 mM CaCh + 0.5 mM KCI + 0. 1 mM MgS0 4 + 0.41 mM Na zHP0 4 + 0.18 mM NaH 2P04 at pH 7.0 was used for th e experiments . Cells were concent rated by circular swinging of the culture dishes whi ch leads to accumulation in th e cent er of the dish. Samples of cells were transferred to the experiment al chamber using a syringe . Exp erimental Chamber. The cha mber (Fig. 1A) corresponds to th at used pre viou sly for Paramecium (perspex; 65 x 110 x 15 mm o uter dimensions; fluid space of 41 x 85 x 1.6 mm) [22].
B
Cell Immobilization and Sedim entation. A sample of cells in experimental solution was exposed for 10 min to 1 mM NiCh dissolved in the same solution. and then transferred to the chamber; 20 more minutes wer eallowed for complete imm obilization of the cells in th e chamber. Measur ements of sedimentation in the vertica l chamber started 30 min after beginni ng of the immobili zation procedure. Recording. Gliding locomotion on the chamber bottom or cover surface was record ed by a CC D-videoca mera (Panasonic F 10, 25 Hz framing rate ) connected to a macro-Jens. Th e resultin g magnification was 26 x . A ring of green light-emitting diod es (565 nm ) attached to the rear of the chamber (Fig. 1B) provided a n even dark -field illumin ati on o f the central area of the chamb er enclosing the cells (Fig. 1A). The ca mera and chamber were fixed to a platform, whi ch was hinged so as to allow a rota tion at 15° intervals betw een the hor izontal (90° inclination ) and vert ical chamber po sition (0°; Fig. 1 B). Cells gliding on th e upp er chamber sur face (right side of cell up) or on the lower chamber sur face (right side of cell down ) were identified by appro priate focu sing of the lens (Fig. 1C). Parameters such as experiment identification, date, time, and ori entation of the cha mber were superimposed o n the video field. Exp erimental Protocol. A sample of 150-200 cells was tr ansferr ed to the chamber with a minimum of agitation. In parti cula r, care was taken to avoid mechani cal disturbances, such as vibrations, during equilibrati on a nd the experi ment , The
~ \
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c Fig. 1. A. Cha mber showing central space for recording of cells in experimental solution (26 x 41 mm ) a nd the lateral areas filled with aga r (1.5% in expo soluti on; dashed a rea ). B. Hinged mount of video reco rding unit (VC) with darkfield illumin ation by a ring of light-emitting diodes (I) behind th e cha mber (C). The unit ca n be rot ated to set the chamber at an y angle from 0 (= verti cal) to 90°C. Definition of inclination angle (8) and identificat ion o f Loxodes inside the chamber with its right side attached either to the low er (L) or upper chamber surface (U). These attachments a re identified by focusing of the macr o-lens on the distal and pro xim al sur face of the chamber, respectively. 0
240 . R. Briiucker, S. Machemcr-Rohnisch, H. Machemer and A. Murakami
Criteria for the Assessment of Steady Gliding Movement. At low oxygen tension, Loxodes commonly glides along a circular track of large radius (Fig. 2). Gliding is occasionally interrupted by spontaneous reversals. The distance between the beginning and the end of a 5-s track ranged between 0.5 and 1.3 mm. Our aim was to evaluate data from undamaged cells showing even gliding motion. We therefore used the start, middle and end points of tracks of little curvature, excluding reversals, for criteria of suitability for evaluation (Fig. 2B-E; see legend for details of selection).
or downward gliding were calculated using the cosine of the angle subtended between the vertical and the chord of a track (see equ. 4 and Fig. 6).
Evaluation of Data. The positions of, and distance between, start and end of a track (= length of cord) were identified. Then, the suitability for further evaluation was tested by means of image analysis (Fig. 2). The rate and orientation of locomotion were determined. From these data, the vertical components of upward
General Behaviour. After 4 hours of incubation in the chamber (experimental space 1.6 ml; horizontal orientation), Loxodes glides on the chamber surfaces at velocities ranging between 150 and 250 um/s. A minor proportion of the cells swims under these conditions. Mechanical shock may induce free swimming along irregular helical courses and/or contraction suggesting mechanosensitivity of the cell. Cells which have been equilibrated for at least one hour and isolated from uncontrolled stimuli such as vibration, heat or strong incident light, continue to show reversals or jerks at irregular intervals. This behaviour resembles spontaneous excitation and ciliary reversal in other ciliates [19, 21, 25]. Observation of cells in the undisturbed experimental solution or in culture fluid, reveals no signs of behavioural responses to gravity comparable to the negative gravitaxis in Paramecium. Direct inspection of tracks recorded with the chamber held in the horizontal and vertical positions do not suggest the existence of directional differences in gliding velocity (Fig. 3).
c
B
A
I D
E
Fig. 2. Criteria for isolation of the steady locomotion rate in Loxodes. Typically, cells glide along straight or slighly curved tracks at constant speed (A). Positions of cells at 3 times separated by the same intervals were determined. For evaluations, the following types of traces were eliminated: B. Traces of large curvature (d ~ w). C. Large changes in gliding velocity. D. Very low rate or unusual form of locomotion suggesting damaged cell. E. Traces indicating that a spontaneous reversal occurred during time of recording.
Statistics. We have chosen nonparametric statistics (calculation of median values; 95% confidence range) because gaussian distribution of data is not assured (see [22]).
Results
Gliding Rates. Quantitative evaluation shows that the gliding rates were virtually the same at all inclinations (Table 1). A small difference exists between the rates on the lower surface (205 um/s) and on the upper surface (231 um/s), which also shows up in the orientational distributions of horizontal rates of locomotion (compare Fig. 4A with B). In Fig. 4A, the horizontal gliding rates of those cells which head toward the agar sides of the chamber (Fig. 1A) are slighly reduced as compared to those moving normal to that axis. It is possible, therefore,
A
A
B
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Fig. 3. Samples of traces of Loxodes with the chamber oriented horizontally (A) and vertically (B). An arrow indicates the direction of gliding from the starting position. In the vertical orientation, cells moved predominantly upward or downward. The traces marked by (',.) have been eliminated from the evaluations according to the criteria shown in Fig. 2.
Grav ity Response in Loxodes . 24 1 Table 1. Median gliding velocities (V, um/s) of Loxodes along planes of different inclinatio n (8; see Fig. 6). A. Cells with their ciliated right side down . B. Cells with their right side up . C. Universal median ra te including all orientation s. Th e median s of uncorrected rat es of all directions on one plane are given together with the 95 % con fidence range (n = num ber of independ ent readings). It is seen that the gliding rates were virt ually independent of the inclination angle suggesting full neut ralizati on of sediment ation
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Fig. 5. Velocity distrib ution s of Lox odes (V-values, Fig. 6) gliding at different inclina tions between 90° (horizontal) and 0° (vert ical) with the right body side down (A) and up (B). Th e top of the polaro grams correspo nds to "up" (except for e = 90°). Medians of the gliding rat es in all l .S'-secrors and at all inclinations are corrected for neutrality toward a non-gravitation al bias (see Fig. 4). Not e that up and down gliding rat es were virt ually unaffected by the inclination angle, or whether the cell was "s itti ng" (A) or " hanging" (B). Figures below the polaro grams are the numb er of velocity readings (n). Inset at lower right gives the velocity calibration of the pola rograms.
242 . R. Braucker, S. Macherner-Rohnisch, H. Machemer and A. Murakami
that the agar blocks established, during equilibration of the cells, minor gradients in aeration (02?, CO 2?) in the recording field. This should, in fact, be seen in horizontal cells, which are assumed to be minimally affected by gravity [22]. In order to offset in the graphical display (Fig. 5) an unknown non-gravitational bias on locomotion, and to allow a comparison of velocity distributions between cells in "sitting" (Fig. SA) and in "hanging" inclination (Fig. 5B), we have normalized the horizontal gliding rates using the universal median of velocities in all directions (206 um/s; n = 12711) for horizontal reference. Correspondingly, correction factors have been employed in Fig. 5 for each of the 24 orientational sectors at 6 angles of inclination (75 to 0 Figure 5 illustrates the overall permanence of the velocity irrespective of the angle of inclination, upward or downward locomotion, and whether the substrate for gliding was below (Loxodes "sitting") or above the cell ("hanging"). With the chamber at very steep inclination (150, 0 0), the rates of upward and downward gliding tended to exceed those occurring perpendicular to this axis. It is possible that errors in identification of gliding cells from those which occasionally separate from the chamber surface and start free swimming have contributed to this minor departure from radial symmetry of the 15 and 0 polarograms (Fig. SA). In depicting essentially the same rates upward and downward, Fig. 5 suggests that Loxodes can compensate effects of gravity on gliding locomotion. Table 1 supplements this conclusion in listing the median velocities at different inclinations of the plane of gliding. 0
0
) .
0
Sedimentation. A simple explanation of the behaviour shown in Fig. 5 might be a neutral buoyancy of Loxodes in the experimental solution. Tests of sedimentation using nickel-immobilized cells established that this assumption does not apply. Loxodes sedimented at a rate of 49 urn/s (Table 2). This value represents the median (+ 7, - 5 um/s; 95% confidence range; n = 312). A similar median value of 47 [!m/s resulted from sedimentations of Loxodes in a different type of chamber (n = 963; unpublished observations). Because sedimentation of freely suspended immobilized cells corresponds to as much as 24% of the gliding rate (median: 206 um/s; Table 1), gravitational pull may affect gliding motion of Loxodes, tending to augment downward displacement and to diminish upward displacement. Determination ofa Gravity-Induced Kinesis. In order to compensate for effects of the gravity force during gliding locomotion as documented in Fig. 5, Loxodes is likely to generate forces equal to those which produce sedimentation, but act in the opposite direction. Irrespective of the actual size of sedimentation during gliding, which cannot be measured, these forces may be represented, under conditions of low Reynolds numbers, in terms of a negative gravikinesis [22].
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Gra vity Respon se in Loxo des . 243
where D and U are the vertical comp onent s of downward and upward gliding velocities, P is the vertical component of the rate of propulsion free of the grav ita tional bias, S is the sediment ation rate , L10 and L1u repre sent the gra vityindu ced downward and upw ard respon ses along the vert ical. Equ ation 3 results from summing of equations 1 and 2 introducing L1 as th e mean of L10 and L1u. For any glid ing track in space, D and U are cosine functions of the indi vidu al declinati on angle (a ) from the fall-line of the chamb er (D', U' ; Fig. 6A) and the inclination (0) of the cha mber (Fig. 6 B):
t = Vi cosa· case , U i = Vi cosa· case,
u
v
A
D
(4) D
wh ere Vi represents the veloc ity of individual gliding . Table 2 shows the median valu es of D' and U', and of D and D, as calculated for 11 specified planes. The velocitie s along the fall-lines, D' and U', are similar for all inclinations, whereas corresponding values of pa irs of D and U (= the median vert ical rat es) decrease with rising inclination (0°: 169 um/s; 75°: near 39 urn/s). Using the sedimentati on rate of 49 ~lm/s and equa tion 3, negat ive med ian values of L1 (i.e., acting inverse to the direction of sedimenta tion) are calcul at ed for each specified plane. These rat es cent er about the value of -49 um/s. Appli cation of equation 3 for the data show n in Fig. 5 suggests an active neutralization of gravity-indu ced forces of sedimentati on of gliding cells. With L1 being defined as the arithmetic mean of L10 and L1 u, how can these subcompo nents be ident ified? Fig. 7 illustrates the notion that, in order to correspo nd to the observed similar downward and upward gliding velocities in any direction (Fig. 5 ), L10 and L1 u must neutralize sedimenta tion, so that L1 =: L1 0 = L1 u. For determinations of L10 and L1u, we assume that the median of gliding in all
B Fig. 7. Scheme to expl ain how ra tes o f upwa rd or down ward gliding (V) in Loxodes can be regulat ed so as to attain a constant value. The grav ity-induced fraction s ( ~1I, ~D ) of th e vertica l compo nents of active propulsion (U, D) a re equal and inverse to S. Thi s ap plies even if the value of S dur ing gliding might differ fro m th at determ ined in the free fluid. A. Upwa rd gliding in arbitra ry dir ection. B. Down ward gliding in arbitrary direction .
direction s (206 + 2, -3 um/s; n = 12711 , Table 1) is a reasonably good app roximat ion of cellular propul sion (P) as unaffected by grav ity. Multiplicati on of P with the cosine of inclination and declinat ion (cos a . costl; equ, 4) then lead s to D and U, respectively, so that, according to
r- -------------------------------~ . .. . . . . . . . . . .... . . . . . • I
,, ,,
I
I
, I
, ,,
,
"
,
"
,
,,
," ,1· · · · · · -_· · ·· ·· · ·· ·· · · · ·
<...... /
A
B
I
,
I
I
/
I
/
I
,r-., ,, "
/U ' •
I
I
"
,, ,,
D~ , " I ,"
,, ,/
v
Fig. 6. Determ inati on of vert ical upward (U) a nd downward (D) gliding rates. A. For gliding on an inclined plan e, th e individual rate (Vi) is tran sformed to U; or D; (= pa rallel to fall-lin e) by mult iplicati o n with th e cosine of the declinat ion angle (a) . B. Side view of (A). The term of U; transforms to U, an d D; to Di , by multiplicat ion with the cosine of the inclination angle (8).
244 . R. Braucker, S. Machemer-Rohnisch, H. Macherner and A. Murakami
equations 1 and 2, Llo and Llu each cancel sedimentation. Discussion Our data show that Loxodes is able to neutralize fully gravitational effects during gliding locomotion. This property of the sensorimotor organization is beneficial to Loxodes, which migrates toward levels of low oxygen tension for food intake and multiplication [8]. Fenchel and Finlay describe locomotion in Loxodes as a random walk, which is modulated by oxygen tension and light intensity [6-9]. To us, it appears to be quite obvious that vertical migrations biased by O 2 and light occur on the precondition that Loxodes manages to offset the disturbance of locomotion due to passive sedimentation. The existence of an inverse strategy has been suggested by Fenchel and Finlay: modulation of gravitaxis of Loxodes (1) by the surrounding O 2 tension (high 02: positive gravitaxis; low 02: negative gravitaxis; Oj-optimum: no gravitaxis; [4,9]), and (2) by light (which shifts the preferred O 2 tension toward even lower concentration levels; [6,8]). More detailed knowledge on the differential effects of gravity, oxygen, and light on gliding, swimming and reversals, respectively, is desirable for full elucidation of the roles of these different stimulus modalities in guiding Loxodes cells to aggregate in their preferred environment.
Terminology. In a previous paper dealing with gravity as affecting locomotion in Paramecium [22], we have called the gravity-induced modulation of active propulsion rates a kinesis, and we tend to apply the same term to the gravity-responses described in this paper. Conventionally, only nondirectional stimuli are thought to induce a kinesis [3,28], whereas we postulate that morphological and physiological organizations of the cell and properties of the stimulus determine a kinetic response in a manner which is difficult to anticipate a priori. Our data suggest that the gravity vector modulates gliding velocity so as to antagonize sedimentation. In this respect, this non-orienting velocity response would resemble a taxis, and we propose to call it a negative gravikinesis. Gravikinesis may be an intermediate between conventional kinesis (nondirectional stimulus, non-orienting response) and taxis (directional stimulus, orienting response). The Problem of Sedimentation during Gliding Motion. In free swimming cells, such as Paramecium, sedimentation directly subtracts from upward swimming, and adds to downward swimming. Locomotion of a cell which glides along a solid substrate may differ from swimming in the unbounded fluid. According to the hydrodynamics of motion at low Reynolds numbers, a self-propelled small body is likely to increase its velocity in the vicinity of a wall due to the unstirred layer of water near a solid boundary (= wall effect); the velocity of an externally driven body of the same size, on the other hand, may decrease near a wall [27,30]. Preponderance in Loxodes of the gliding rate (206 um/sl.over the swimming rate (70-180 um/s; [4])
agrees with hydrodynamic predictions. It also follows that our value of passive sedimentation (S = 49 um/s), which was determined with the immobilized cells freely suspended, might not fully apply to gliding cells, where some reduction of sedimentation would be expected. It is easily seen from Fig. 7 that, with our observations of constant gliding velocities (Fig. 5), a reduced sedimentation implies a decrease of the gravikinetic response by the same amount. In the extreme and unlikely case that S = 0, both Llo and Llu would be reduced to zero to comply with the observations. The rate of sedimentation during gliding remains unknown for principal reasons. Irrespective of the sedimentation component in a gliding cell, the effective gravitational force (downward) needs to be cancelled by an active counterforce (upward). With the density of Loxodes cells ranging between 1.01 and 1.02 glml (preliminary, unpublished data), the pressure of the cytoplasm on the lower membrane of a vertically oriented cell is in the order of 0.03 Pa (or 0.3 dyne/cm-). This gravitational force is neutralized by an extra kinetic effort of the cilia, which may be expressed in terms of an increment (or decrement) in rate of locomotion, which the same ciliary work is able to generate. The existence of a gravikinetic response in Loxodes, albeit invisible in gliding under natural gravity, is further supported by observations during centrifugation showing that, with rising gravity and increasing rates of sedimentation, the upward gliding rates were unchanged, whereas the downward gliding rates continuously increased (unpublished data).
Possible Electrophysiological Implications. Until now, Loxodes has not been investigated electrophysiologically. The values of LlD and Llu are so far unrelated to sites and functions of sensory transduction. In Paramecium, Stylonychia, Stentor and Didinium, the anterior soma membrane bears depolarizing mechanoreceptor channels [2, 11, 24, 29]. Paramecium and Stylonychia, in addition, show hyperpolarizing mechanoreceptor responses following stimulation of the posterior soma. Small depolarizations from the resting potential depress forward locomotion, hyperpolarizations augment swimming in Paramecium [21,23]. With determinations of vertical and horizontal swimming rates, and of sedimentation, at hand in Paramecium, it has been argued that the gravitational load of the cytoplasm on the lower (= anterior) membrane depolarizes downward-swimming cells, and hyperpolarizes the lower (= posterior) membrane in cells swimming upward [22]. Our calculation of the value and sign of Llo and Llu, agrees with the electrophysiologically sound hypothesis that Loxodes generates a steady positive-going gravireceptor potential during downward gliding, and a negative-going gravireceptor potential during upward gliding. Loxodes bears statocystoids (" Muller vesicles") near the anteriorventral cell end, and these organelles have been implicated in gravisensation theories [4,5]. Assuming, in accordance with the statocyst hypothesis of gravisensation [13-15], that gravity-induced deformation of a mechanosensory substrate constitutes the adequate stimulus, the statocystoids of Loxodes, which enclose a statolith of
Gravity Response in Loxodes . 245 BaS04 [10,26], may have a crucial function in gravisensory transduction [5]. More conventional transduction principles like stretch deformation of the anterior and posterior soma membrane, analogous to mechanotransduction in Paramecium, may be envisioned as well. Physiological probing into Loxodes including the roles of the Muller vesicles, and detailed behavioural studies are desirable to help unveiling the mechanism of gravitransduction in this cell. Acknowledgements This work was supported by the Bundesminister fur Forschung und Technologie, Federal Republic of Germany, grant QV 8857 (to HM), and the Special Fund for Promoting Science and Technology, STA, Japan (to AM). We thank P. F. M. Teunis for critical discussions and G. Krumbach, U. Schilken and P. Ullrich for devoted technical assistance.
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Key words: Mechanosensation - Gravitaxis - Loxodes Richard Brauker, Arbeitsgruppe Zellulare Erregungsphysiologie, Fakulrat fiir Biologie, Ruhr-Universitat, D-4630 Bochum 1, FRG