J. PROTOZOOL. 24(3),394-101 (1977).

Geotactic Behavior of Chlamydomonas I k p a t tinent

of

BARRY BEAN Biology, Leliigh Uniz~ersity,Bethleheni, Pennsylvania

SYNOPSIS. The normal negative geotaxis (motility oriented against gravity) of Chlamydomonas is an energy-dependent response that requires coordinated flagellar activity. It is evident from quantitative assays that the rate of geotaxis is steady, and slow relative to the average swimndng speed. Geotaxis is inhibited when the horizontal swimming path is less than 200 pm, suggesting that normal geotactic reorientation maneuvers involve long gradual turns. Videomicrographic tracking of cells confirms that such turns are common. I n contrast, contact-reorientations generate random cell orientations. When collision frequencies increase, geotaxis in inhibited. The mechanism of normal geotactic orientation. then, depends on long slow reorientation maneuvers (from net downward to net upward vectors) that require hundreds of micrometers of free swiniming spare. Mechanisms of geotaxis that would require passive reorientation or sedimentation. or rapid active responding. are excluded. Unusually dense populations sediment with atypical rapidity, probably due to formation of functionally aggregated subpopulations. Sodium azide causes an inhibition of orientation hehaiior that is selective relative to its effects on general motility. Evidenc:e presented suggests that active physiologic mechanisms for geotaxis should be reconsidered. Index Key Words: Chlamyduniunas : geotaxis : orientation behavior; pattern swimming; videotracking.

THE

geotactic behavior of microorganisms has received occasional attention from scientists of various disciplines since the \ k t o r i a n period. Many niici-oorganisms iincluding Ch [antsdomonos) from divers,. taxonomic groups typically exhibit negatiw gcotaxis-motility orientcd against the gravitational field 12, 16, 20, 21, 25, 26, 31, 10). T h c phenomenon of negative geotaxis is particularly intriguing since the organisms are often of higher density than their usual surroundings; to achieve geotactic redistribution microorganisms must expend physiologic energy in coordinated motile beha\.ior. W’hilc the single term gcotaris has been widely applied t o microbial orientation behavior, it is conceivable that different mcchanisms of geotactic responding may be employed by diffcrent species. T h e methods ust,d for studying geotaxis in microorganisms have been largely nonquantitativc,, involving cither ( a ) qualitative or semi-quantitative evaluation of responding a t the populational level, or ( b ; microscopic obsei-\.ation of small populations or of individual organisms ha\.ing orientcd movement. There has been little understanding of thc mechanism ( s ) used by microorganisms in orienting t o gravity. Many possibilities have been considered? including mechanisms that operate by virtue of hydrodynamic properties of the cell as it interacts either pssively or actively Lvith its surroundings. Purely passive physical processes (e.g. the mode of passive sedimentation of cells) are not sufficient to account for hchavior that qualifies as true geotaxis (16, 25, 31, 40’). Kuznicki (25) found no support for passive mechanisms in his studies on the sedimentation of immobilized Parawicciunz, and concluded “the question of the mechanism of spacv orientation of protozoa in gravity fields remains still open.” Roberts ( 3 1 ) accounts for geotaxis by consideration of cell shape and sedimentation hydrodynamics; this statement also has shortcomings as mplained by N’inet & Jahn ( 4 0 ) . The gravity-propulsion model of LVitiet & Jahn (20, 40) explains geotactic reorientation as thc physical consequence of a torque produced on actively swimming cells that have an asymhape and a gyrational s\c.irnniing path. IVhile this model uing, and pcrhaps applicable to many species, it presuppost’s a set of physical and ph).siologic restrictions. T h e authors have not ytst sho\vn that thv qualifications required by their model do in fact rnrrclate appropriately \vith the positiw, negative, or zero geotactic rc’sponwcs obccrved in various types of frre-s\viniminc cells. In reccnt years there has not heen significant reconsideration of the possible existence (in at least some cases) of geotaxis

mechanisms of a more behavioral nature. Recent studies on other experimental systems of microbial behavior suggest that, in a t lcast some instances, rrsponding involves integrated physiologic processing a t receptor, transmission, and effector levels of organization. This organization appears true for bacterial chemotasis ( 1 ) , for chemotaxis and other responses in Paramecium ( 2 3 ) , and for phototaxis in Chlamydomonas (17, 35-37) among others ( 7 ) . There has also been some reccnt discussion concerning the reception of gravity signals in higher plants-the possibility still exists that statolith systems are not solely responsible for gravity susception in geotropism (14). In vicw of these developments, and considering the structural and functional diversity of groups among the microorganisms, and recognizing that several geotactic behavior mechanisms might exist, it tvould seem \vise to consider possible physiologic mechanisms also for geotaxis. T h e studics prcsented here were undertaken in the hope that simple quantitative methods might permit understanding of the geotaxis mechanisms. A simple quantitative method has been deviscd to describe the geotactic redistribution of cells in population5 of Chlaniydomonas. A quantitative characterization of the geotactic response of Chlamydomonas reinhardtii is presented, and some mechanistic implications are discussed. ,4lOIlg n i t h microorganisms from several other groups, the chlaniydonionads received the attention of some early behavioral biologists who, during the period 1880-1920, described pattern s\viinining and responses to light, temperature, gravity, and chemicals (10, 21, 21, 26, 27, 29, 3 9 ) . Some aspects of the intriguing phototactic behavior of Chlnmydomonas have been studied recently in some detail (5, 6, 11, 12, 17, 18, 33-37), but the mechanisms of phototaxis remain largely unexplained. I n nndertaking studies on a 2nd system of orientation behavior (geotaxis) in this organism, opportunities for understanding of both response systems are expanded. M A T E R I A L S A N D METHODS

Organisms and Culture Conditions.-Chlamydomonas reinhnrdfii strain 137c, mating types plus and minus, and the mutant pfl (icolated by R. A. Lewin) were obtained from M. R. Kifkin. T h e strains 21GR and certain phototactic mutants (PT series) thereof \\ere generously provided by R. Hirschberg a n d R. L. Stavis before publication of their results (17, 36). Neg-1, -2, and -3, phototaxis-defective mutants ( 1 8 ) of strain 137+, were sup-

3 94

GEOTAXIS IN Chlamydomonas plied by G. Hudock. Strains y-1 (32) and cw15 ( 9 ) were provided by P. Siekevitz. Strain cw92 (19) was obtained from J. Hyams. The stigma-deficient mutant ey (15) was provided by R. D. Smyth. Liquid cultures were routinely grown in medium containing the salts mixture ( K,HP04, KH,P04, NH4C1, CaC12, MgS04) of )/2 HSM (38); supplemented with a trace metals solution modified from that of Jones ( 2 2 ) by substitution of equimolar trace metal concentrations of CuSO, for CuC1, and of NaMo04 4 ; supplemented with a final concentrafor (NH,) 6 M ~ 7 0 L 'also tion of 0.001% (w/v) FeC1,-6H2O. Dilute phosphate buffer, a mixture of K2HP04 and KH2P04 equivalent to that normally contained in the growth medium, was used for washing and resuspension of cells, and, after filtration (Millipore HA filters, Millipore Corp., Bedford, MA) as diluent and electrolyte for the Coulter Counter. Cultures were maintained at 25 & 0.5 C, under a 12-hr light-dark cycle of illumination with cool-white fluorescent lamps (light phase beginning at 6:OO A.M. Eastern time). For dark grown cultures, growth medium was supplemented with sterile CH3COONa to a final concentration of 0.2% (w/v). For experiments involving cell concentration as an experimental variable, cells from cultures in the exponential phase of growth were concentrated by phototactic aggregation to values of -5 X 107 cells/ml, and diluted to the desired final cell densities. For experiments involving azide inhibition, highly motile cells were twice washed by gentle centrifugation and resuspended in buffer at a final concentration of 7.7 X 105 cells/ml. Subcultures were supplemented with freshly prepared stock solutions of NaN, to give the desired final inhibitor concentrations. Measurement of Swimming Speed.-Rates of motility for suspensions of Chlamydomonas have been quantitatively evaluated by the method of Ojakian & Katz (28). The number of cells passing through a defined area in a hemocytometer within a measured period were used to calculate a population-average swimming speed. Values determined by this method were compatible with those derived from videomicrographic tracking and from direct timing of individual cells under the microscope (after correction for the proportion of nonmotile cells). Evaluation of Phototactic Responding.-For most experiments described here, nonquantitative but sensitive assays for phototaxis have been used. Samples of cultures to be tested were sealed with clean modeling clay (Milton-Bradley or an autoclavable nontoxic equivalent) in capillary tubes (Kimax, 100 m m ) . Three kinds of observations were made: ( a ) cross-tube phototaxis, ( b ) short phototopotaxis, and ( c ) running phototaxis. Cross-tube Phototaxis.-Capillary tube cultures were studied against a black background on the stage of a dissecting microscope. Tubes were illuminated with white light from a position roughly in the horizontal plane of the stage, and nearly perpendicular to the long axis of the tube. Photoresponsive cells accumulated in a band along one wall of the capillary tube, and, when the tube was quickly rolled for 180", photoresponsive cells rapidly reoriented and swarmed directly to the opposite side of the tube. Normal motile cultures characteristically had positive phototaxis and a rapid uniform reorientation response. Short Phototopotaxis.-The capillary sample was turned so that illumination was aligned with the long axis of the tube to give cells an unimpeded path relative to the light source. Brief examination permitted evaluation of the direction, speed, and uniformity of responding in the test population. Running Phototaxis.-Capillary-tube cultures in the horizontal

395

position were exposed to a steady illumination from a direction aligned with the long axis of the tube for 10-30 min. After exposure, control and experimental preparations were compared by microscopic examination. For comparison of phototaxis rates, it is sometimes desirable to begin the assay with cells already concentrated at one end-cells of most genotypes can be SO distributed by preliminary phototactic or geotactic accumulation. While this introduces a bias against nonresponsive cells, these represent only a small fraction of normal uninhibited populations. I n some experiments, an alternative method for quantitative evaluation of phototaxis has been used. In this method the hemocytometer motility assay (28) was modified by introduction of actinic light source. The procedure contrasts undirectional motility values for the absence and presence of actinic light. The resulting data can be expressed either as a phototaxis rate, in absolute pm/sec of net phototactic movement, or as a phototaxis coefficient by relation to the population-average motility (Bean & Purvis, unpublished results). Population Level Geotaxis Assays.-Each quantitative assay for geotaxis was performed by placing a sample of a homogeneous suspension of cells into a capillary tube and allowing the cells to redistribute themselves in the tube during subsequent constant-temperature incubation in the dark for a measured period. Capillary tubes of uniform bore, usually 100 pl, 116 mm long (Drummond microcaps, 100 p l ) , or 100-mm melting point capillaries (Kimax) , were used. The tubes were scored prior to filling by etching the glass at the midpoint and at points 3 mm from either end. After loading the tube with a sample of the suspension of motile cells, the tube was quickly sealed at both ends with modeling clay to give a closed, bubble-free chamber with a 3-mm clay plug at each end. The tube was immediately placed in vertical position in a light-proof container at constant temperature (22 C ) , and the time was recorded. Care was taken to avoid exposure of loaded tubes to light. After the desired period of dark incubation, usually 20 to 90 min, the tube was broken at the etched mark; the time was recorded. The number of cells in each half-tube was then determined; the cell numbers, the length of the half-tube (i.e. the distance from the clay plug to the midpoint of the capillary tube), and the period of the assay (i.e. the time during which the tube remained in the upright position), were recorded for quantification of geotaxis, described below. Cell numbers were usually determined by use of a Coulter Counter, model ZB (Coulter Electronics, Hialeah, F L ) after appropriate dilution into particle-free potassium phosphate buffer. Cell numbers, or relative numbers of cells, were also determined by counting of clones on agar, hemocytometer counting, or scintillation counting of cells appropriately prelabeled with radioactive NaHCO, or CH,COONa. The following formula was used for quantification of geotaxis:

1. where N, = the number of cells in the top half of the assay tube after incubation for the time t; N,, = the number of cells in the bottom half of the tube after incubation for time t; and N, = (Nb Nt)/2 is the half-tube population. G, is a dimensionless ratio expressing the proportion of the half-tube population that has undergone net redistribution between half-chambers during the period of the assay. The ratio takes on values from -1 to +1, where the sign corresponds to the direction of movement, negative values reflecting net upward movement of cells in the capillary tube. The absolute value of

+

3 96

GEOTASISI N Chlamydontonas

G, increases as a function of the duration of the assay, t, as expected (see Results). A quantitative expression for the rate of geotactic movement is : 3 -.

G,d Gd=t

where d = length ( i n p i ) of the half-chambcr (i.e. brt\veen the score marks at the midpoint and the clay plug at the end of the assay tube), t = the period (in hec) of the assay (i.e. duration in the vertical position:. G , then has rate units of positive or negative pni/sec, which can be directly contrasted tvith swimming speed. l'ideomicrographic Recorditig.-Cell suspensions \\-ere studied in frmh wet mounts under conditions that permitted cells to be free-swimming. For studics on gcoraxis, saniplcs of highly motile culturrs of the strain neg-2 (or other nonp'lototactic genotypes) were loaded into small capillary chambers (0.05 mm path length "microslidcs," l'itro Dynaniics, Rockaway, NJ, or similar chambers), and mounted in the vertical position on a micromanipulator. This specimen chamber was illuminated from behind with red light [ 10-30 \Y tungston incandesccnt illumination passed through a N e t t #66 filter). The behavior of cells in thc c~xperiiiiental tubes was recorded ivith the aid of a dissecting microscope at 90X optical magnification using a \ideorecording system (Sony .AL\\'C 1400 videocamera, or the equivalent, and Sony AY3650 videotaperecorder). Distance calibration of the video image. and final magnification were determined by comparison \vith the recorded image of ii micrometer scale. Data rccordcd ivith this system were displayed for analysis at normal or rcduced tape speed, and on a frame-by-frame hasis. The swimming pathu of cdls \\.ere summariled by the following method. Taped sequences to be used for analysis xverc chosen at random. A transparent sheet (28 x 43 c m ) was centered and attached to the screcn (39 X 49 cm) of the video monitor, and thc successive positions of a l l cells included Ivithin that area tvere transpowd to that sheet. In various experiments, positions mnrkcd were those corresponding to a fixcd number of frames in the videorecording; data \\.ere recorded at 30 frames/scc in all experiments, and positions were transposed at intervals of 6. 7, or 10 frames in various playback analyses. Thus, the actual horizontal and vertical distances, as well as the elapsed time are represented in the arparation of points on the summary sheets, but information on changes in depth Ivithin the focd plane of the microscope is lost in this summary trcatment. Some distance aberration is present for points a.c\-ayfrom the center of the screen, reaching a maximum of -10% expansion of the lincar scale at the perimeter of thc transparent shect relative to the scale at the (,enterof the scrcen. Summar)- tracks of fair numbers of randomly aelrcted motile cells, representing their movement through several seconds of real tiinr., can be readily contrasted and/or used ;I< a basis for statistical analysis. This summary method permits characterization of thc quality and quantity of various movements that occur in thc population. including information about .\\vininiing spccd and direction a s \\-ell as the character and frequency of changes in speed and direction. RESULTS

Gr nc~ral C'haractciiJtirr o f Geotczzis.-The geotactic response of population\ of C h l n t n ~ d o m o n aha, ~ 11wn quantified by the tnpillary tube a5,a'r \)\tein and Coulter counting. G, values for

such cultures typically fall in the range -4 to -8 pm/sec. Multiple samples from a single culture usually give G, values that agree within & l pm/sec. G, values for samples from different cultures vary more widely, probably reflecting variation in physiologic characteristics such as: ( a ) population density; ( b j relative numbers of nonmotile cells; ( c ) relative adhesiveness of cells; ( d ) variation in the population-average swimming speed; ( e ) time of day and/or time within the division cycle, etc. The "typical" values given here apply to cultures that have: ( a ) a population-average motility of 60 -C 20 ym/sec (reported as approximate mean 2 S.D.; timed rates for individual cells may be as high as 120 pm/sec); ( b ) not less than 80% motile cells (as ascertained by microscopy) ; and ( c ) low adhesiveness in relation to the duration of the geotaxis assay (i.e. the average time that a cell is immobilized on glass surfaces is less than I/lo of the assay period). There is no evidence of a difference in adhesiveness between the top and bottom halves of a capillary tube. Kinetics of Geotczris.-Typical results of an experiment in which several assay tubes were prepared from a single culture and sampled in sequence during a 2-hr period, as shown in Fig. 1. G, values remain essentially constant through this period, while the value of G, increases with an essentially linear relationship to time. The 2-hr period is clearly sufficient for a great proportion of the motile cells to migrate to the top half of a 100-mni capillary tube. Dependence of Gcotaxis on Ccll Concentration.-Typical results of studies on the rclationship between geotactic responding and the concentration of cells are shown in Fig. 2. For experiments of this type, exponential growth phase cells are first concentrated to densities above 107 cells/ml. The dramatic phototactic aggregation that these cells exhibit was exploited as a preferred method for concentrating cells rapidly and with a minimum of damage or loss of motility. At high cell concentrations therc is an inhibition of the normal negative geotactic response. Negative geotaxis was not observed in samples with initial honiogencous population densities above lo7 cells/ml, and, in populations of 3 X 10' cells/ml and above, positive geotaxis \\.as noted (cells \ \ w e redistributed toward the bottom of the tube). I t is of interest that the rate of sedimentation of cells in these dense populations was rapid. Rates (G,) abovc + l o p i / s e c reflect sedimentation that is faster than typical rates for negative geotaxis; these rates are also faster than those for passivc sedimentation that are characteristic of individual nonmotile rclls at lower concentrations. Control Studies and Energy Requirement.-No significant pattern of net distribution to % the capillary was seen in populations kept in the dark but in a perfectly horizontal position, even after hours of incubation. Moreover, under these conditions, as under those previously reported ( 4 2 ) , cells initially collected at one end of a capillary tube (by centrifugation, phototaxis, or geotaxis) ivould redistribute themselves randomly in the tube. I t has been sholvn in control experiments of several kinds that geotaxis is an energy-requiring cellular activity. I n these experiments, the behavior of mutant or experimentally treated cells has been contrasted with that of normal cells. The normal negative geotactic response was abscnt ( i x . G, > 0) in cells that ( a ) have been freshly killed by fixation with formaldehyde, glutaraldehyde. iodine, or other agents; ( b ) have lost or withdraivn their flagella spontaneously, as occurs before cell division; ( c ) have been experimentally deflagellated, by adsorption to a mixed cellulose acetate-cellulose nitrate filter (Millipore Corp., Hcdfo~cl, bftl, t)pc MF) or by chemical method7 ( 4 1 ) ; ( d ) cariy the pfl mutation (for paralyzed flagella); ( e ) have been

397

GEOTAXIS IN Chlamydomonas

+ I5

-1.0

+ I0

;

- .8

t5

.3m (3'

E

x

/

o -5

-.6

-10

+

a

I

I

I

I

3x105

lo6

3x106

lo7

I

I

3x107 7 . 2 ~ 1 0 ~

C e l l concentration c e l Is/m I

- .4

Fig. 2. Dependence of the geotactic response on cell concentration. Symbols X, 0, and 0 represent cells derived from independent starting cultures. Each data point represents the average of 4 determinations that were run simultaneously, except for 0 , which was a single determination.

-.2

0 0

30

60 90 minutes

120

Fig. 1. Kinetics of geotaxis. Samples of a culture with 1.7 x 10" cells/ml were assayed as described in Materials and Methods. treated with chemical inhibitors that impair motility, such as NiCl,, CdC1, antimycin A, EDTA, or NaN,; and ( f ) have been deprived of light for extended periods, and have become nonmotile. After geotactic accumulation of a population of healthy cells at the top of a capillary tube, the tube may be inverted, and the cells migrate at their normal geotactic rate toward the opposite (upward) end of the tube. This process may be repeated several times, in the dark, without depleting the energy supply of the cells. After prolonged incubation in the dark, however, cells become nonmotile, and fail to exhibit normal geotaxis. I t is important to point out a number of conditions that do not interfere with normal geotactic reorientation. For instance, photosynthetic activity is not required for geotaxis. Cells of the genotype y-1 can be dark-grown in the presence of acetate without development of the normal capacity for photosynthesis. Under such growth conditions, large numbers of nonmotile cells occur, and quantification of geotaxis is not meaningful. It is evident from microscopic examination of the assay tubes prepared with these cultures that motile cells perform the geotactic response at an apparently normal rate. Similar results hold for dark-grown wild-type cells. A number of mutant strains, some after long periods of cultivation independent of their parental wild-type C . reinhardtii strains, have quite normal rates of geotaxis. These strains include several nonphototactic mutants (although some mutants isolated as nonphototactic mutants also show abnormal geotaxis) ; G, values for some of these mutants are presented in Table 1. Other mutant strains that have normal geotaxis rates include: PT9, PT11, neg-1, neg-2, neg-3, ey, cw15, cw92 and others ( 3 ) . Under certain conditions, the inhibitory actions of sodium azide and of nickel ions on motility can be reversed by Ca++,with restoration of the ability to perform geotaxis.

Azide Inhibition of Geotaxis.-Stavis (35, 36) has reported that sodium azide causes a selective inhibition of phototaxis in C. reinhardtii. The effects on geotaxis in Chlarnydomonas by different concentrations of this inhibitor are summarized in Fig. 3. Phototaxis and general motility, as well as geotaxis, were monitored in a series of cultures containing concentrations of 0 to 1 mM sodium azide. Increase in azide concentrations resulted in a progressive inhibition of geotaxis. I t was ascertained by microscopic examinations of such cultures that the elevation of azide concentration was also accompanied by parallel increases in inhibition of general motility and of phototaxis (in qualitative assays). There may be a slight selectivity for inhibition of phototaxis relative to geotaxis, and of geotaxis relative to effects on general motility, but any such selectivity is not pronounced or persistent. Furthermore, there are timedependent changes in the effects of azidc inhibition as observed by these methods. The exposure to 350 PM NaN3, at early times (e.g. 25 min after exposure to the inhibitor), appears to have little effect on general motility, but causes a clear (but not complete) inhibition of the rate of geotaxis, and an essentially complete inhibition of phototopotaxis. These results for early times after exposure to inhibitor are consistent with those of Stavis (35, 36). At later times (after 60 min exposure to 350 piu NaN,) a partial inhibition of general motility is apparent, but motile cells do respond to light. Inhibition of Chlamydomonas by moderate concentrations of azide produces a mixed population of cells that have varying degrees and combinations of the following characteristics : no apparent inhibition, blebbing or ballooning of the flagellar membrane, abnormal swimming style, loss of flagellar coordination, flagellar paralysis, and breakage or loss of one or both flagella. Inhibited cells can recover from these effects to regain full motility; the recovery process also varies from cell to cell. Effects of Capillary T u b e Radius on Geotaxis.-A series of experiments was performed to assess the influence of the bore of the capillary tube on the rate of geotactic responding. The results of one experiment are summarized in Fig. 4. In this and all other experiments of this series, increasingly negative rates of geotaxis were associated with larger capillary tube radius; restriction of tube radius interfered with the normal capacity for upward orientation. Cell Tracking and Videomicrographic Analysis.-To study the

398

GEOTAX~S IN Chlawiydomonas

TABLE 1. C,I r~uluesfor C:. reinhardtii itrain 21GR and Aome its phototaxis mutants *

___

__________

Strain

21GR PT2 PT5 PTlO

* Cultures

-

of

-1.2

r

G,i (pni 'sec) t

._.

-6 5 . -6 6, -60. -6 6.

-7 5 -6 5 -65 -7 4

of 21GR and the phototactic mutant strains PT2, PT5, and PTlO were cultured in parallel. t Values for duplicate determinations of G,i

-.8

*-

-.4

0

niotilc lwhavior of individnal cellu o r small populations of Chlaniydonionas microscopically during geotaxis, it is desirable to niinimiie the normally strong phototactic response. A combination of 2 experimental conditions \vas exploited to eliminate the latter. First, nonphototactic mutants neg-2 or P T 9 ivere used. Thesf, mutants exhibit normal sivimming style and speed (17, 18, 361, but are nonresponsi\,e to light sources that elicit orientrd motility in wild-type strains of ChlainTdomonas. Second, specimens tvere illuminatd with red light (see Materials and M[.thods-. Under these circumstances geotaxis occurs at the normal rate. It \vas evident from videorrcorded data that the apparent swinimin~s p e d s for cells follotving diffcrcnt vrctor paths \\('re essentially the same‘; for cells on straight paths, those heading upward, dou.n\vard or hori7ontally had equal average ratrs of movement. Furthermore, thcre \vas little or no passive rrorientation of freely siispended cells during any momentary cessation of active propulsive movement. Cells typically did not turn or tumble during periods of passi\,e sedimentation. Several types of cell reorientation are observed in geotactic populations. One type is the result of physical interaction of the rcll with another object, usually another crll or the xvalls of the chambrr. Such directional changes may occur w-ith or Xvithout a stop or intcrruption in the forivard propulsion of the crll. As c x p e c t d , stops occur most frequmtly in conjunction lvith head-on collisions (i.e. a t angles 45-90" between the swimnliiig vector and the contact \urfac(,', and less frequently with glancing angle interactions. IVhrn an interruption docs occur, it niay include a brief period (-0.1-0.5 sec) during jvhich the cvll spins in place, sivims in tight circles, or sivims in reverse for some distance before hmding off in a new direction with a normal swimming style. The frequency of intel.action-reorientations is a function of the population density. size of the experimental chamber, presence of inrrt matter, etc. .lnalysis of videorecordings indicates that, to at least a first approximation, iiiteraction-reorientations occur Ivith equal frequency among the subpopulations \vith net upxvarcl or net downivard motility vwtors, and that the final angle for recovery \rectors is essentially random. A 2nd typc of reorientation that occurs at loiv frequency in nonpliotoresponsive ci3lls involves an apparently spontaneous angular turn in sivimming direction, again, u i t h or without an accompanying stop. Due to their low frequency, and to difficulty in distinguishing angular turns from interaction-reorientations undcr the conditions used in these qualitative studies, the importance of spontancous angular turns remains an open question. This type of turn is more frcqurnt in photoresponsi\.e cultures. Photoresponsive cclls exhibit a stop and angular turn in response to suddcn incrcasrs in light intensit!.. .\ 3rd type of reorientation mancuver is a smooth gradual continuous lmv-anglc turn that takw plact. during nortnal swimming over distances of hundreds of micromcters. This kind of

+ .4 40

0

Fig. 3.

80 minutes

120

160

Inhibition of geotaxis by sodium azide.

turn can be noted for cells oriented in any direction, and often results in 20-50" angle changes in the swimming vector of the cell during the 3.3 sec period commonly used in cell-tracking experiments. Such turns are common in cultures of nonphotoresponsib e Chlamydomonas. In populations with low collision frequencies, thi.; is a frequent reorientation maneuver. T h e impact of such turns for understanding the mechanism of geotaxis niay be great. DISCUSSION The normal negative geotactic response of C. ieinhaidtii occurs at a rate that is characteristically steady and slow. Cells move up the tube \vith an essentially constant net average rate (Gd) over substantial periods of time. For the experiment shown in Fig. 1, this rate ( G d ) approximates -6 to -7 p i / s e c net upward movement. By contrast, typical cultures have population-average swimming speeds of 50 to 70 pm/sec. Geotaxis, then, is the consequence of a constant small bias in the net propulsive effort in thc upward direction. Geotaxis is an energy-requiring behavior of Chlnmydomonas. T h e response requires coordinated flagellar activity, as shown in cxperinients in ivhich flagella are removed or their activity

-8.0

+4.0

r

'

0

1

I

I

I

I

200

400

600

800

1000

tube r a d i u s in m i c r o m e t e r s

Fig. 4. The influence of capillary tube radius on geotaxis. x 10' cells/ml were assayed in quadruplicate using capillary tubes with each of the 4 different bore radii indicated. Samples of a strain 137' culture containing 1.6

GEOTAXIS IN Chlamydomonas restricted by chemical inhibition, genetic paralysis, or energy limitation. Geotaxis is independent of the immediate influence of light, i.e. nonphotosynthetic or nonphototactic cells can exhibit normal geotaxis. The negative geotactic response is lost along with motility after prolonged starvation. Conditions that restrict motility, even though reversible, inhibit negative geotaxis, and often cause slow sedimentation of the nonmotile cells (G, = + I to +3 pm/sec). The achievement of the upward orientation of motility requires a horizontal free swimming path of several hundred micrometers. Two lines of evidence support this view. When the space available for horizontal swimming is experimentally restricted in capillary tubes of small diameter, net upward migration is restricted. These results suggest that the pertinent type of reorientation maneuvering, by which Chlamydomonas orients upward, requires horizontal space on the order of hundreds of micrometers. Videomicrographic recording experiments confirm that long, slow turns of this magnitude are common in populations of Chlamydomonas that are performing geotaxis. Substantiating evidence comes from experiments in which population density was varied. At high cell concentrations, normal negative geotactic orientation is inhibited, and for concentrations up to -lo7 cells/ml, the degree of inhibition increases as a function of cell concentration. This inhibition is probably due to the increase in frequency of collisions between cells, which decreases the mean free swimming path of the individual cells. Increase in collisions, and the consequent relative increase in interaction-reorientations, inhibits upward orientation. These observations provide definitive evidence that neither passive reorientation nor interaction-reorientations plays a role in achievement of the normal negative orientation to gravity. For high density populations, the mean free path ( A ) of cells can be approximated by the relationship from kinetic theory,

3.

A=-----

1 27rd2N’

where d is the particle diameter, and N is the particle concentration. For Chlamydomonas with an approximate average diameter of 8 pm, concentrations of lo7 and 3 X 107 cells/ml correspond to mean free path values of -350 p m and 117 pm, respectively. Data presented in Fig. 2, again reveal significant inhibition of gmtartic orientation when the free swimming path of cells is less than a few hundred micrometers. Unusual positive geotaxis values are seen at unusually high population densities. At cell concentrations above 3 X 107 cells/ ml, mean free paths are restricted to less than 100 pm. Since each cell requires an appreciable volume of swimming space to maintain motility, and since there is some lag following collisions before each cell can resume normal swimming, it seems likely that nonrandom aggregations of cells would occur in high density populations. According to Stokes’ Law, aggregations should sediment passively a t rates faster than those characteristic of individual cells, generating the observed high positive rates of sedimentation. Another contrast between the 2 space-restriction experiments confirms this interpretation. Increase in population density causes restriction in the mean free swimming path in all directions. By contrast, limiting the radius of a vertical capillary tube (with length of the various tubes held constant) primarily generates restriction of swimming in near-horizontal paths. By restriction of the horizontal paths only, the formation of celI aggregations is not dramatic. Distribution of cells in the tube remains relatively random because long slow turns from net

399

downward orientation to net upward orientation, and vice versa, cannot occur. This is consistent with observations reported here -positive geotaxis values are not observed for narrow tubes at moderate cell densities, but are observed even in wide tubes at high cell densities. Experiments on the inhibition of geotaxis by sodium wide are intriguing. Azide treatment inhibits geotaxis, phototaxis, and motility with different, although somewhat variable, selectivity. Stavis (35, 36) reported that phototaxis was quite effectively inhibited before the onset of general inhibition of motility, and the same relationship may hold for geotaxis. Longer periods, however, are required for measurement of geotaxis than of phototaxis, and the degree of inhibition of motility caused by azide under the conditions used here was significant. If some selectivity for inhibition of geotaxis is granted, then the site of inhibition may be a process at the transmission level of behavioral signaling that is common to phototaxis and geotaxis. If this type of effect can be definitively demonstrated for azide or for any other inhibitor, then geotaxis must occur by a physiologic mechanism. The alternative remains, however, that azide causes a selective inhibition of motility at the effector level. I n Chlamydomonas, several swimming strokes and styles, modes of turning, etc., can be observed; at low concentration azide may influence certain aspects of motility, while at higher concentrations severe general inhibition of motility results from inhibition at several targets. The effects of azide on flagellar structure and function are heterogeneous, suggesting that multiple targets in the flagellar apparatus may be affected. Stavis (35, 36) suggested that azide treatment of Chlamydomonas may result in depolarization of the electrochemical gradient across a cellular membrane. If so, then a membrane-localized electrochemical signaling system may be involved in the normal coordination of stroking between the 2 flagella of each cell. While the data presented here are not incompatible with the mechanisms of geotaxis proposed by Winet & Jahn (401, it is not clear that Chlamydomonas exhibits all of the characteristics required by that hypothesis. The Winet & Jahn model would require that a gyratory swimming path be involved in geotactic reorientation. In addition, there is no good evidence to rule out mechanisms that involve physiologic activities including receptor, transmission, and effector levels of information processing, Further studies on the physiology and genetics of geotaxis should resohe these mechanistic issues. I t is striking that the patterns of geotactic migration in relation to tube width reported here for Chlamydomonas are quite the opposite of those reported by Brinkmann ( 4 ) for Euglena. Brinkmann noted the distribution of Euglena (initial concentration 106 cells/ml) in long (36 cm) sealed tubes after 15-hr incubation in the dark ( 4 ) . He stated that in wide tubes Euglena accumulate on the bottom, while in narrower tubes (with diameters in the range of the mean free path calculated by Brinkmann) cells accumulate at the top ( 4 ) . He concluded that geotaxis does not occur in Euglena, and that net downward migration is due to sedimentation, while net upward migration is due to special hydrodynamic restrictions imposed by the capillary tube ( 4 ) . Brinkmann’s failure to observe negative geotaxis in wide tubes under this set of conditions constitutes negative evidence, which in itself cannot be considered conclusive. Control studies, including tube-inversion experiments, observations on migration in progress, details of cytologic observations, consideration of changes in the physiology and composition of cells and their medium during incubation, etc., were not reported. Brinkmann

400

GEOTAXISI S Chlamydomonas

did not report rcsults on capillary tubes of diameter less than 0.9 mm. In grncral, then, results dwcribed here arc very different from his. In both large tubvs and in capillary tubes that do not restrict the mean frcc path, healthy motile Chlapnydomonas migrate upward; when the niean free path is limited by size of the capillary, upward migration is inhibited. Thus, whether or not Brinkmann's nicthods, analysis, or interpretations arc accepted, Chlamydomonar presents a distinct casc. The character of geotaxis describcd here would aid in the survival of Chfarnydomonas relativc to thr presumed selectivc pressures encountered during its evolution. Photosynthetic freshwater or soil organisms that are heavier than water, confronted with darkness and/or transient loss of their motile apparatus, reproduce most effectively only if they can rcturn to the surface. T h e active behavior described hcrc, including both randomizing thigniotaxis (inrrraction-reorientationsthat en;lblc cells to iiiove around obstacles) and slow steady negative protasis, along with positive phototaxis. would optimize the probability that a cell could find its way to the surface. Cells that havc these behavior patterns rcproduce rfft~tively,and cells that do not are subject to strong, inmediate selection. Thc rapid sediiiientation in dmse populations of Chlamydomonas is consistent with roles for geotaxis, aggregate formation. and passive sedimentation that have bcrn implicated in the rncchanism of pattern swiiiiniing in other microorganisms (8, 13, 30). Chlamydomonas also has stable pattern swimming under appropriate conditions (39). T h e observations and mcthods described here for analysis of geotasis and for characterbation of scdinicntation niipht be useful in further analysis of microbial pattern swimming. The availability of bchavioral mutants and the opportunity for genetic analysis makes C h l a t n y domonas an attractive subject for such studies. Evaluation of physical, physiologic, and grnetic variablrz niiylit distinguish the relative influenccs of geotasis, phototasis, and pattern swimming in thc reproductivc. succcss of this spccics.

ACKR'O\\'I.ED~El,lENTS

I am gratrful to Drs. Roria Hirschbere and Robert L. Stavis for \timrtlating 3 i i d aqwn rrrhniiev of idmv. .ind for providiiir! niutant strain? brfore publication of their findines (17, 3G), and to Prof. Donald R. Griffin for his grnrrosity and mcourage.erncnt. Also appreciated are thc intcwsr and help of: George Bays. Alan Harris, Dorothy R. Hartman, David Kaean. Peter Klose, Xlarqarct M. Krau iec, Steven Krau iec. John lf JIi7e1, Da\ id .4. Nusblatt, and Mac E. P u n k REFERENCES 1. Adler J. 1973. Chemotaxis in Escherichia coli. in PerezMiravete A, ed., Behaviour of Microorganisms. Plenum Press, New York, pp. 1-15. 2. Bean B. 1975. Geotaxis in Chlanipdomonas. J. Cell Biol. 67, 24a. y. -.1976. Flagellar coordination in Chlamydomonas can be analyzed by studying geotaxis in behavioral mutants. Genetics 83. S5-6. 4. Brinkmann K. 1968. Keinc Geotaxis bei Euglena. 2. Pflanzenphysiol. 59, 12-6. 5 . Bruce VG. 1970. The biological clock in Chlamydomonas reinhardi. J. Protozoology 17, 328-34. 6. - 1972. Mutants of the biological clock in Chlamydomonar reinhnrdi. Genetics 70, 537-48. 7. Carlile MJ. 1975. Taxes and tropisms: diversity. biological significance and evolution: in Carlile M J, ed.. Primitice Sensory and Communication Systems: The Taxes arid Ttopisnir of Microorganisms and Cells: Academic Press, London. 1-28.

8. Childress WS, Levandovsky M, Spiegel EA. 1975. Nonlinear solutions of equations describing bioronvection, in Wu TY, Brakaw CJ, Brenncn C, eds., Swimming and Flying in Nature, Plenum Press, New York, 1, 361-75. 9. Davies DR, Plaskitt A. 1971. Genetical and structural analyses of cell-wall formation in Chlamydomonas reinhardi. Genet. Res. Camb. 17. 33-43. 10. Desroche P. '1912. Rdactions de Chlamydomonas aux Agents Physiques. Schultz, Paris. 11. Feinleib ME. Currv GM. 1967. Methods for measuring phototaxis of cell ' popuiations and individual cells. Physiol Plantarum 20, 1083-95. 12. -- 1971. The relationship between stimulus intensity and driented phototactic response (topotaxis) in Chlamydomonac. Physiol. Plantarum 25, 346-52. 13. Gittleson SM, Jahn TL. 1968. Pattern swimming by Polytomella agilis. .4m. Nat. 102, 413-25. 14. Gordon SA, Cohen MJ, eds., 1971. Gravify and T h e Organism. U. of Chicago Press, Chicago. 15. Hartshorne JN. 1953. The function of the eyespot in Chlampdomonas. New Phytol. 52, 292-7. 16. Haupt W. 1962. Geotaxk, in Ruhland W,ed., Handbuch der Pflanzenphysiologie, Springer-Verlag, Berlin, l7/2,390-5. 17. Hirschberg R. Stavis R. 1977. Phototaxis mutants of Chlantydomonas reinhardtii. J. Bacteriol. 129, 803-8. 18. Hudock GA, Hudock MO. 1973. Phototaxis: isolation of mutant strains of Chlamydomonas reinhardi with reversed sign of response. 1. Protozool. 20, 139-40. 19. Hyams J. Davies DR. 1972. The induction and characterization of cell wall mutants of Chlamydomonas reinhardi. Mutat. Res. 14,381-9. 20. Jahn TL, Votta JJ. 1972. Locomotion of Protozoa. Ann. Reo. Fluid Mech. 4,93-116. 21. Jennings HS. 1906. Behavior of the Lower Organisms. Columbia University Press, New York. 22. Jones RF. 1962. Extracellular mucilage of the red alga Porphyridium cruentam. J. Cell. Comp. Phy.rio1. 50, 61-4. 23. Kung C, Chang SY, Satow Y, Van Houten J, Hansnia H. 1975. Genetic dissection of behavior in Paramecium. Science 188, 898-90+. 24. Kuwada Y. 1916. Some peculiarities observed in the culture of Chlamydomonas. Bot. Mag. ( T o k y o ) 30, 347-58. 25. Kuzhicki L. 1968. Behavior of Paramecium in gravity fields. I. Sinking of immobilized specimens. Acta Protorool. 5, 109-1 7. 26. Massart J. 1891. Recherches sur les organismes infkrieurs ( I ) . Acad. R . Sci. Lett. Beaux-arts Belg. 22, 148-67. 27. Mast SO. 1911. Light and T h e Behauior of Organisms. M'iley, Kew York. 28. Ojakian GK, K;itz UE. 1955. A simpk tcchnique for the measurement of swimming speeds of Chlamydomonas. Exp. Cell Res. 81.487-91. 29. Pfeffer W. 1888. Ober chemotaktische Bewegungen von Baktericn, Flagellaten und Volvocineen. Unters. Bot. Znst. Tubingen 2, 482-661. 30. Plesset MS, Whipple CG, Winet H. 1975. Analysis of the steady state of the bioconvection in swarms of swimming microorganisins, in Wu TY, Brokaw CJ, Brennen C , eds., Swimming and F I y i q in Nature, Plenum Press, New York, 1, 339-60. 31. Roberts AM. 1974. Geotaxk in motile microorgariisms. J. Exp. Biol. 53, 687-99. 32. Sager R, Palade GE. 1954. Chloroplast structure in green and yellow strains of Chlamydomonas. Ex$. Cell RRS. 7 , 58i-8. 33. Smyth RD. 1971. Genetic Control of Phototactic Aggregation in Chlamydomonas reinhardtii. Doctoral dissertation, Lrniv, Calif. a t Los Angeles. Univ. Microfilms No. 72-11, 899. 34. -, Ebersold WT. 1970. A Chlamydomonas mutant with altered phototactic response. Genetics 64,562. 35. Stavis RL. 1974. The effect of a i d e on phototaxis in Chlampdomonas reinhardi. Proc. Natl. Acad. Sci., U.S.A. 71, 1824-7. 36. 1974. Phototaxis in Chlamydomonas: A Sensory Receptor System. Doctoral dissertation, Albert Einstein College of Medicine, New York. University Microfilms NO. 75-27,884. 37. -, Hirschberg R. 1973. Phototaxis in Chlamydomonas reinhardtii. J . Cell Biol. 59, 367-77. 38. Sueoka N, Chiang KS, Kates JR. 1967. Deoxyribonucleic acid replication in meiosis of Chlamydomonas reinhardi. I.

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GEOTAXIS IN Chlamydomonas Isotopic transfer experiments with a strain producing eight zoospores. J. Mol. Biol. 25,47-66. 39. Wager H. 1910. On the effect of gravity upon the movements and aggregation of Euglena viridis, Ehrb., and other microorganisms. Phil. Trans. R , SOC.London B201. 333-90. 70. Whet H, Jahn TL. 1974. Geotaxis'in protozoa. I. A

propulsion-gravity model for Tetrahymena (Ciliata) . 1. Theor. Biol. 46, 449-65. 41. Witman GB, Carlson K, Berliner J, Rosenbaum JL. 1972. Chlamydomonas flagella. 1. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes. J. Cell Biol. 54,407-39.

J. PROTOZOOL. 24( 3 ) , 401-405 (1977).

Effects of Neurochemicals upon a Dinoflagellate Photoresponse" RICHARD B. FORWARD, JR. Duke University Marine Laboratory, Beaufort, North Carolina 28.516 and Zoology Department, Duke University, Durham, North Carolina 27706

SYNOPSIS. Photoresponsiveness by Gymnodinium splendens Lebour was monitored quantitatively by a microscope-television system. Exposure to the catecholamines DOPA and Dopamine caused a decrease in light sensitivity, while 0.01 mM norepinephrine, epinephrine, or isoproterenol did not affect photoresponsiveness. Classical catecholamine blocking agents, dichloroisoproterenol, propranolol, and dibenzyline, and an inhibitor of DOPA synthesis, a-methyl-p-tyrosine, caused an increase in sensitivity. I n addition, acetylcholine and an inhibitor of acetylcholinesterase activity, eserine, caused an increase in sensitivity, while an inhibitor of acetylcholine action atropine, had the opposite effect. These experiments suggest that G . splendens may have an antagonistic catecholamine-cholinergic system which participates in regulating photosensitivity.

Index Key Words: Gymnodinium splendens; dinoflagellate; phototaxis; catecholamines; acetylcholine.

E

VIDENCE exists for the presence of choline and catecholamine substances in dinoflagellates. A chemical resembling acetylcholine has been extracted from G y m n o d i n i u m veneficum Ballantine ( 1 ) and Amphidiniurn carteri Hulburt (26, 29). In more detailed studies, however, A. carteri was found to contain acrylylcholine, choline o-sulfate and an unidentified choline ester (20, 25). Furthermore, a substance resembling norepinephrine was found in Noctiluca ( 2 3 ) . Since neurochemicals appear to be present in dinoflagellates, an important question concerns their functional significance. Some investigators have suggested that neurochemicals are responsible for the toxicity of these protozoa for animals. While available evidence ( c g . Refs. 1, 26) certainly suggests that this is true, these substances may also be involved in dinoflagellate sensory systems, perhaps in ways analogous to their use in animaI nervous-sensory systems. This suggestion is supported in part by recent work implicating catecholamines and choline compounds in chemosensory responses by the heterotrophic marine dinoflagellate Crypthecodinium cahnii ( 12, 13). Most dinoflagellates display phototaxis upon stimulation with a directional light source. At high Iight intensities, the behavioral response sequence for the marine species G y m n o d i n i u m splendens Lebour consists of an initial cessation of movement (stopresponse) followed by swimming in the direction of the stimulus beam. The action spectra for both responses are identical, having maxima at 450 and 280 nm ( 9 ) , which is typical of dinoflagellates ( 11 ) . The present study was undertaken to investigate possible neurochemical involvcment in behavioral responses to light by G. splendens. Several criteria were established to determine whether this in fact occurs; photoresponsiveness must be predictably altered by ( a ) neurochemicals, ( b ) substances which mimic neurochemical action, ( c ) substances which block neurochemical action and ( d ) substances which affect neurochemical metabolism. In addition, the presence of neurochemicals within

* This investigation was supported by Research Grant GB-90885, from the National Science Foundation.

the cells should be determined. This report deals with results of experiments in which photobehavior was altered by the external addition of neurochemicals and other substances. MATERIALS AND METHODS G y m n o d i n i u m splendens was cultivated as described previously ( 9 ) on a light-dark cycle of 12: 12 hr a t 20 C. Since cells grown under these conditions have a circadian rhythm in photoresponsiveness ( 9 ) , all tests were begun during the time when they were maximally responsive, i.e. 2 to 3 hr after the beginning of the light phase. Individual cultures were grown in -125 nil of mcdium and tested when cell densities reached -22000 cells/ ml. Although the G . splendens culture was not bacteria-free, bacterial growth was minimized by using sterile glassware and culture medium. The selection of drugs used in this study was based upon classical drug action in mammalian systems. Altrration in photoresponsiveness was measured upon systematically exposing the cells to catecholamines L-epinephrine bitartrate, L-norepinephrine HCI, Dopamine, and L-DOPA; to a drug which mimics catrcholamine activity m-isoproterenol HCI; to drugs which block their action dichloroisoproterenol HCl, L-propranolol HCl, D-propranolol HC1 and dibenzyline; and to a drug which inhibits synthesis, DL-a-methyl-p-tyrosine. Similar tests were run with the cholinergic compound acetylcholine chloride and substances which inhibit its action (atropine sulfate) or which affects its metabolism (eserine sulfate). All chemicals were obtained from Sigma Chemical Go. except D- and L-propranolol (Ayrrst Laboratories, Montreal, Canada) and Dibenzyline ( SmithKline Laboratories). All solutions containing the test chemicals were prepared in fresh culture medium on the day of experimentation, except for dibenzyline, which was first dissolved in 0.2 ml of absolute ethanol and then added to the medium. The general procedure was to subdivide individual cultures into 6 parts, each of which was placed in a 50-ml beaker. To 5 of these subsamples were added various concentrations of the chemicals so that the final