J. Eukarjor. Microhiol., 46(5), 1999 pp. 532-541 0 1999 by the Society of Protozoologists

Warm Microgradients Elicit Adaptive Behavior in Isotropically Cooled, Inert Populations of Oxytrichu bifaria (Ciliophora, Hypotrichida)

FILIPPO BARBANERA, FABRIZIO ERRA and NICOLA RICCI Dipartimento di Etologia, Ecologia ed Evoluzione, via A. Volta, 6-56126 Pisa, Italy

ABSTRACT. In order to investigate the physiological potentialities of behaviorally inert Oxytricha bifaria, cooled from 24 to 9" C according to an already standardized protocol, a warm microgradient was created in the experimental chamber and the behavior of ciliates was analyzed both at the level of the passing warm wave front (dynamic microgradient), and, afterwards, when the thermal gradient stabilized (static microgradient). We monitored the general behavior of the experimental populations by means of (i) their centroid, (ii) the ethograms of single oxytrichas, and (iii) calculating the numerical indices and rates of their creeping tracks. It was found that (a) the population moves towards the heating source, (b) the oxytrichas react immediately to the thermal stimulus, (c) creeping forwards (d) at very high velocity (e) along uninterrupted looping tracks ( f ) according to precise mechanisms of positivehegative orthokinesis, thus orientating towards the environmental optimum. Moreover, (8) the ciliates accumulate in the warmest area, correcting their creeping by means of many specific behavioral patterns (the Side Stepping Reaction) once the gradient is stabilized. At 9" C, despite their inertness, the ciliates are still able to behave adaptively reacting immediately and orientatedly, once a directional factor (the thermal gradient) arises in an isotropic environment.

Key Words. Ethogram, klinokinesis, low temperature, orthokinesis. thermoaccumulation.

HE behavior of the ciliate 0. bifaria (Ciliophora, Hypotri- T chida) at progressively lower temperatures, isotropically applied (i.e. without thermal gradients) according to a cooling protocol, had been studied previously (Ricci, Barbanera and Erra 1998a). The general mobility of these experimental pop- ulations decreased with the temperature, to its lowest value at 9" C, when the oxytrichas are completely inert. Their very poor locomotion occurs mainly backwards, at a very low velocity and it is interrupted by relatively long-lasting stops. In order to analyze quantitatively such a reduced mobility, several new in- dices and rates were proposed and used to describe precisely the very wrinkled tracks performed by ciliates at low temper- atures (Ricci, Barbanera and Erra 1998b). These results seemed directly related to the cooling kinetics (Inoue and Nakaoka 1990; Machemer and Teunis 1996), and to the related progres- sive decrease in the metabolic economy of the cells, which is responsible for both the reduction of the velocity of the organ- isms (Glaser 1924), and the different states of membrane po- tential (Connolly et al. 1985; Hildebrand 1978; Martinac and Machemer 1984). The almost complete absence of mobility, however, represents quite an uncommon state for protozoa. In- cessant movement in time and in space is the principal char- acteristic of life, so that it assumes a fundamental survival value also for small living entities such as the protozoa (Carlson 1962; Fenchel 1987; Jahn and Bovee 1967; Sollberger 1962). On the other hand, the behavior exhibited by the oxytrichas at 9" C cannot be considered an aberrant one (Kittredge 1980) since (a) they show a prompt recovery on return to 24" C and (b) no organism died in the cooled droplet (Ricci, Barbanera and Erra 1998a). The behavior of oxytrichas at 9" C, however reduced, seems to be the expression of still perfectly physio- logical internal conditions: is it still adaptive? In other words, can it still express locomotory outputs capable of increasing the survival probabilities of the cooled oxytrichas (Meyer and Guil- lot 1990)? For these reasons we decided to carry out a series of experiments on the basis of Mendelssohn's pioneering works (Mendelssohn 1895, 1902a. b, c), which demonstrated that pro- tozoa placed in a thermal gradient accumulate in regions with temperatures close to their culturing temperatures, called the optimum region (Jennings 1904, 1906). Tawada and Oosawa (1972), as well as other authors (Matsuoka, Mamiya and Taneda 1990; Nakaoka and Oosawa 1977), reported that swimming protozoa increase their forward velocity when moving towards their optimum region, while they exhibit frequent changes in

Corresponding Author: E Barbanera-Telephone number: 0039-50- 500840; FAX number: 0039-50-24653; Email: nic.ric@discat.unipi.it

swimming direction when moving away from that region. Ta- wada and Miyamoto (1973) found that Paramecium exhibits thermotaxis when the rate of change of temperature is greater than 0.055' CIS.

This paper deals with the results of the experiments we car- ried out by applying a warm microgradient in the uniform en- vironment experimentally kept at 9" C, namely in an environ- ment lacking any directional information possibly useful to search for more favorable conditions. Such a warm microgra- dient represented a directional stimulus acting on the organisms, at first, as a passing warm wave front (1st phase: dynamic mi- crogradient) and, then, once the thermodynamic equilibrium with the cooling apparatus is reached, as a stabilized gradient (2nd phase: static microgradient). Differently from the exam- ples reported above (Tawada and Oosawa 1972), we chose to apply a warm microgradient in an already conditioned environ- ment, so that the cooled organisms during the 1st phase expe- rienced the temperature rise in both space and in time (at a certain fixed distance from the heating source), and, during the 2nd phase, the temperature rise only in space (moving towards the heating source).

MATERIALS AND METHODS The TAB 1 strain of 0. bifariu (Ciliophora, Hypotrichida)

grown at 24" C was used throughout our experiments. Cultures were grown according to an already standardized protocol (Ric- ci, Banchetti and Cetera 1980). Experimental populations (from one-day-starved cultures) were transferred into a droplet (z 150 p.1) placed on a glass slide in a Petri dish. This played the role of a small moist chamber: this experimental setup was isotrop- ically thermally-conditioned by means of a CRIOTERM 10-80 thermostat. The protocol we followed in order to uniformly cool the experimental populations down to 9" C was the same de- scribed for the previous experiments (Ricci, Barbanera and Erra 1998a): however, the use of the new high-sensitive thermometer acquired to describe exactly the thermal gradient (see below), enabled us to measure the actual temperature of the experimen- tal populations with a degree of precision ten times greater than that used before. It was found that the so called 9" C popula- tions, actually, are at 8.2" C: this value is reported throughout this paper. Whenever our 8.2" C populations are to be compared with the equivalent populations of the previous experiments, the latter are cited as 9" C populations.

To create a thermal gradient, a capillary tube (outer diam.: 1.4 mm; inner diam.: 1.2 mm) was placed between the ther- mally-conditioned chamber and the slide with the experimental populations (Fig. 1): a water flow of 1.2 mYs at 24 2 1" C

532

BARBANERA ET AL.-THERMAL GRADIENTS AND ADAPTIVE BEHAVIOR 533

B Y

rh 1

A

d

3 4 5

#3 #2 #1 CA

capillary tube

/ I 23 A 11

Fig. 1. Schematic representation of the experimental set-up, as seen from above (upper left scheme) and in cross section (lower left scheme). 1: thermally-conditioned experimental chamber; 2: the Petri dish cover; 3: the experimental glass slide; 4: the experimental droplet; 5: the experimental field; 6: the control glass slide used to measure the actual trend of temperature; A -+ A': the thermostat water flow, conditioning the experimental chamber at 8.2" C; B + B': the capillary tube water flow originating the warm microgradient. The panel on the right shows the enlargement of 5 as seen from above (upper scheme) and in cross section (lower scheme): CA, # 1, # 2 and # 3 indicate the areas studied within in the experimental field.

passed through the capillary tube upon the opening of a stop- cock, thus creating the warm gradient.

The TV recordings refer to an area of 8.9 mm . 5.8 mm divided into four parts: the first (called CA-area) in correspon- dence of the capillary tube measures 8.1 mm2 (1.4 mm . 5.8 mm), and the other three, measuring 14.5 rnm* each (2.5 mm . 5.8 mm), were called 1-, 2- and 3-area respectively according to their progressively increasing distance from the CA-area it- self.

The thermal gradient in the experimental populations was monitored both in space (CA-, 1-, 2-, and 3-areas) and in time

(every 10 s) by a digital thermometer (Delta OHM, HD 9218) with a TCK bulb (diam. = 1 mm, sensitivity: 2 0.1" C), spe- cifically calibrated by the Standard Bureau of ENEL (Italian Electricity Board) to work in the range of temperature used throughout our experiments. The real spatial and temporal trend of the temperatures (Fig. 2) was measured in the control glass slide (Fig. 1 : lower glass slide, 6) and found to be definitely the same as that in the experimental glass slide (Fig. 1: upper glass slide, 3). Figure 2 shows that after about two minutes the warm microgradient is already static: the temperature drops from the capillary tube (16.5" C: namely the value resulting

534 J. EUKARYOT. MICROBIOL., VOL. 46, NO. 5, SEPTEMBER-OCTOBER 1999

3 - area

120- 160s 17

2 - area I - area CA - area

Fig. 2. The spatial (abscissas: the different areas of the experimental field, see 5 in Fig. 1) and the temporal trend (on the right of the curves: time, s) of the temperature, shown on the ordinates, have been measured every 10 s.

from the 8.2" C imposed by the thermostat and the 24" C of the water flowing in the capillary tube) to the I-area (14.9' C), to the 2-area (12.4" C) and to the 3-area (10.3' C), so that the thermal gradient measures about 0.9" C/mm on average. The temperature, in the parts of the experimental droplet not TV- recorded, decreases progressively from 10.3 to 8.2" C.

Large experimental populations (120 organisms) were used to study the trend of their general distribution in time, while smaller populations (70 organisms) were used to study the lo- comotory patterns of the single organisms. To describe the gen- eral mobility of the large populations, the centroid of the dis- tribution of the organisms in the experimental field was calcu- lated every 10 s as the mean of their positions and reported onto a Cartesian coordinates system. From the TV recordings of the smaller populations, several ethograms (Eibl-Eibesfeldt 1967) were drawn: (a) at 24" C, (b) at 9" C, (c) at the level of the warm wave front (defined as a temperature increase of 0.2" C/s, calculated from the data of Fig. 2), and (d) in the CA-area where the behavior was analyzed for the first five minutes.

Due to the complexity of the model system we investigated (in turn depending upon both the technical difficulties and the biological answer, namely the behavior), after studying it thor- oughly by means of many, repeated experimental sessions each devoted to the analysis of the different aspects of the phenom- enon itself, we decided to run a final, general experiment in order to present and to discuss a unique set of experimental data encompassing all the different aspects to monitor. This choice was made for the sake of the clarity, because it enabled us (a) to deal with real, homogeneous data, all referring to the same experimental populations in the same experiment, (b) to reduce their variance. No significant difference ever occurred between different sets of data referring to the same aspect of the phenomenon studied.

A synopsis of the basic concepts of the ethogram of 0. bi- faria (Ricci 1981) needs to be given here in order to establish the context in which the ethographical findings we are reporting in this paper may be understood. Oxytricha bifaria moves on the substrate creeping along leftward arcs (A-) and linear seg- ments (S), two of the three Long Lasting Elements (LLE) gen- erally described for the ethogram of ciliated protozoa (Ricci 1990). These arcs are characterized by several parameters: a) their radius (r, pm); b) their central angle (p, degrees); c) their total length (I, pm); d) the velocity of the ciliates traveling along them (u, p d s ) ; e) their duration in time (At, s). The SSR (Side Stepping Reaction), one of the four Short Lasting Ele-

/

I, = L/t ( p d s )

R, = D/t ( p d s ) Fig. 3. The basic elements of a track traveled by a ciliate from A

to B: L. its real length; D, the distance between A and B; t, the time spent to travel from A to B along L. On the right the kinetic index (Ik), the geometric index (IJ, and the rate of displacement (Rd) are shown together with the way of calculating them.

ments (SLE) described for the creeping tracks of ciliated pro- tozoa (Ricci 1996), was also measured: we studied its correc- tion angle (a. degrees) and its Backward Motion (BM), de- scribed by its total length (I, pm), by the velocity of ciliate traveling along it (v, p d s ) and by its duration in time (At, s). At 9" C we described also the pSSR (prolonged Side Stepping Reaction) adding the values of the radius (r, pm) and of the central angle (p, degrees) for its BM performed as a leftward arc (Ricci, Barbanera and Erra 1998a). Our sets of data have been statistically processed both to have their statistical descrip- tion (.t 2 SD, namely the mean value and the relative standard deviation) and to make inferences about possible differences occurring among them by means of the Kruskal-Wallis non- parametric test. A significant difference is characterized by 0.01 < p < 0.05, while a highly significant difference by "p" values smaller than 0.01.

Finally, the movement of a creeping ciliate along a track of a certain length (L), traveled in a time (t), and with the extremes lying at a distance D (Fig. 3) is described exhaustively by means of two indices and one rate (Ricci, Barbanera and Erra 1998b): (1) the kinetic index (I, = L/t), namely the average velocity ( p d s ) , in a certain way measuring the action of "cil- iary engines"; (2) the geometric index (I, = D L ) describing the straightness of the track by means of a dimensionless num- ber and similarly measuring the state of the "ciliary steering wheel" (0 5 I, 5 1); and (3) the displacement rate (Rd = D/ t), which integrates the first two indices and expresses their combined effect on the track by a unique measure (pds) , which, in turn, defines the average displacement rate or, in other words, the effectiveness of the track in displacing the organism in space.

RESULTS The analysis of our data led us to consider two main groups

of results: the first [A] deals with the description of the general mobility of the population (Fig. 4-6), the second [B] with the behavior of the single oxytrichas for a detailed description and measurement of their locomotion, in the presence of both the warm wave front (B. I, Fig. 7, 8) and the static microgradients (B. 11, Fig. 9).

A: the general mobility of the population. The oxytrichas at first progressively move towards the heating source signifi-

BARB ANERA ET AL.-THERMAL GRADIENTS AND ADAPTIVE BEHAVIOR 535

3.0 r 1 T

1.5

0.0

I I

120- 160 s 1

r T - r

I 3-area I 2-area ~ I -area j CA - area

Fig. 4. The kinetics of the cell densities (ordinates: number of cells per square millimeter) in the space (abscissas: 3-area, 2-area, I-area and CA-area) and in time (as indicated by the four bars for each area) after the onset of the thermal gradient. The cell densities were measured every 10 s in each 40 s period of time (upper left inset): each bar expresses the mean value of the four measurements for each 40 s period, the standard deviation being represented by the vertical segment. The cell density in each area was measured also before the onset of the thermal gradient (controls), and the differences among the four values were found to be statistically not significant (p = 0.13). The average value (0.48 2 0.08) is indicated by the horizontal dotted line.

cantly accumulating into the CA-area in less than three minutes from the onset of the thermal gradient (black bars in Fig. 4: p = 0.004). Such a thermoaccumulation is described by the pro- gressive movement of the centroid of the population towards the CA-area (Fig. 5: A curve), in turn following the positive thermal gradient (Fig. 5: B curve): from the initial 8.2" C in the 3-area to the final 16.5" C in the CA-area in less than three minutes. The movement of the centroid of the population every 40 s (Fig. 6: from 8.2 to 9.5 to 11.2 to 13.9 to 16.5" C: see the lower scheme for the trend of thermal gradient), shows that the population moves towards the highest temperature at increas- ingly higher velocities, from 17 p d s along AB (about 5 mm from the CA-area), to 35.4 p d s along BC (4.5 mm from the CA-area), to 56.1 p d s along CD (3.5 mm from the CA-area) and to 59.9pds along DE (2.1 mm from the CA-area). If we consider the component of the velocity of the centroid parallel to the thermal gradient, its value increases from 7 . 2 p d s (AB) to 29 p d s (BC), to 36.1 p d s (CD), and finally to 59.8 p d s (DE). As for the acute angle (y) lying between the vector con- necting two successive positions of the track and its component parallel to the thermal gradient, its value generally decreases from 65" (AB) to 35" (BC), to 50" (CD), and finally to 5" (DE).

B. The behavior of single oxytrichas. B. I : the response to the warm wave front (dynamic microgradient). Three examples of typical tracks performed by the oxytrichas when the wave front reaches the I-area, the 2-area and finally the 3-area, are shown in Fig. 7. The organisms react with the same kind of response, regardless of the area where they are: they immedi- ately (a) creep forwards, (b) at a very high velocity, (c) without any interrupting SSR. In the I-area (Fig. 7: panel I: I-area) the warm wave front reaches the organism in only 4 s (Fig. 7: panel 11: velocity of wave front: 690 p d s ) , as revealed by the posi- tion of the arrowhead (corresponding to the opening of the wa- ter flow in the capillary tube), which is very close to the black arrow (corresponding to the arrival of the warm wave front onto the organism). The ciliate, in turn, starts creeping immediately, as shown by the white arrow very close to the black one (time

16

h

Y

B k

14 - r

12 E

10

" 8 20 40 60 80 100 120 140 160

Time (s )

Fig. 5. The trend in time (abscissas, s) of the distance between the centroid of the population and the capillary tube (on the left or- dinates, A curve: D-D) is shown in comparison with the tempera- tures (on the right ordinates) actually occurring at the level of the centroid itself (B curve: 0-0).

delay: 1 s) at a very high velocity (Fig. 8: 1-area) along a coiled trajectory, consisting of 2-3 almost geometrically perfect circles whose centers, generally, are progressively displaced to- wards the capillary tube: this reaction in its complete form, has been called the "looping pattern". The organisms in I-area stop reacting with the looping pattern when they are so close to the warmest region that they reach the CA-area (Fig. 7, Fig. 8: *). In the 2-area (Fig. 7 and Fig. 8: 2-area) and in the 3-area (Fig. 7 and Fig. 8: 3-area) the arrival of the warm wave front is delayed by 8 s and 35 s, respectively, due to the progressive

165OC

gradient 8 2 ° C

.B e 1 - 0

0 " " ~ 1 ' " ' ' ~ ' ~ ' ~

gradient 8 2 ° C

* 1 - 0

0 " " ~ 1 ' " ' ' ~ ' ~ ' ~ 0 1 2 3 4 5 6 7 8 9

Abscissa of centroid X lo3 ( pm )

I 3 -area I 2-area 1 1 -area 1 area(

Fig. 6. The successive positions (A, B, C, D, E) of the centroid of the population, calculated every 40 s (compare with Fig. 4) in the ex- perimental field (schematically represented in the lower panel) consid- ered as a Cartesian coordinate system. The vectors (bold lines) uniting the successive positions indicate the relative velocities of the centroid (inset: V) to move from a position to the next one. The vectors (thin lines) parallel to the abscissas indicate the component (inset: V,) of the same velocities parallel to the direction of the thermal gradient, whose trend is given in the lower scheme. The angle between each bold vector and its relative thin vector is indicated as y.

536

E l

El 190 p d s

J. EUKARYOT. MICROBIOL., VOL. 46, NO. 5 , SEPTEMBER-OCTOBER 1999

310 p d s 690 p d s

5

3 -area

\

CA area

2 -area 1 -area

\

SSR

J

I,=210 p d s I, = 0.13 Rd=28 p d S

I,=353 p d s I, =0.12 %=43 p d s

I,=280 I J d S

I, = 0.13

Rd=38 P d S

decrease in its velocity (Fig. 7: panel 11: 310 p d s and 190 pm/ s, respectively) coupled with the obvious greater distance to travel: the response to the increasing temperature is always im- mediate, as shown by the white arrows, very close to the black ones. At the end of their looping patterns both the organisms of the 2-area and of the 3-area have to travel a certain distance to arrive in the CA-area, where they start performing a series of SSR in correspondence with the borderline between the CA- and the 1-area.

The looping pattern can be described thoroughly by the in- dices reported in Fig. 7, panel 111: the increased I, (8.2" C: 117

* 25 p d s , n = 10; 1-area: 280 p d s ; 2-area: 353 p d s ; 3- area: 210 p d s ) are balanced by the significant reduction of I, (8.2" C: 0.30 +- 0.17, n = 10; 1-area: 0.13; 2-area: 0.12; 3-area: 0.13) so that the average dispersal of the oxytrichas, measured by their Rd is very small (8.2" C: 34 2 21pn/s, n = 10; 1-area: 38 p d s ; 2-area: 43 p d s ; 3-area: 28 p d s ) . The analysis of the instantaneous velocity along the tracks of Fig. 7 is shown in Fig. 8: its sudden increase at the arrival of the warm wave front (black arrow) is evident beyond any doubt. The interesting finding is the periodic variations of the instantaneous velocity, very clear in the track of the 2-area. The organism accelerates

800

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BARBANERA ET AL.-THERMAL GRADIENTS AND ADAPTIVE BEHAVIOR

200

0 6

-400

800

600 400 200

0

600 400

-

-

-200 0

-400 1 , . , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , I 0 10 20 30 40 50 60 70 80

Time ( s )

Fig. 8. The trend in time (abscissas, s) of the instantaneous velocity (ordinates, w d s ) along the three tracks of Fig. 7. The indications are the same as in Fig. 7.

when it creeps towards the wanner areas (Fig. 8: 2-area: from the white arrow to 1, from 2 to 3, from 4 to 5) , while it decel- erates when it moves away from them (Fig. 8: 2-area: from 1 to 2, from 3 to 4, from 5 to 6). After entering the CA-area (Fig. 8: 1-area, *), the organisms creep very slowly (G 100 p d s ) for a while (2 min), and start performing several SSR in cor- respondence with the borders of the CA-area itself (Fig. 7).

As the temperature drops from 24 to 9" C, the ethogram changes as shown in Table 1, according to the results reported in the previous paper (Ricci, Barbanera and Erra 1998a). At the arrival of the warm wave front a series of counter-changes oc- curs in comparison with the 9" C (isotropic condition): (a) the A- is performed more frequently (93% vs 55%); (b) its radius does not vary; (c) its central angle increases very significantly (113" vs 73"); (d) its length increases very significantly (1,170 vs 603 pm); (e) the velocity of the oxytricha creeping along it increases very significantly (361 vs 118 p d s ) ; ( f ) its duration in time decreases significantly (5.2 s vs 3.8 s); (8) the SSR disappear completely, like the pSSR (Table 1).

B. 11: The response to the stabilized warm microgradient (static microgradient). As reported in Fig. 2, the thermal gra-

SSR,

SSR' I 1 - area

537

...'..'...'. '... '.. ' 16.5" C

14.8" C . . . . . , . . . , . . . . . . . . . . . . . . . . . . . . . . . .

CA - area

Fig. 9. The track of an oxytricha maneuvering slightly (average velocity: = 130 pmls) towards the CA-area performing a series of SSR (SSR,, . . . SSR,). The lower scheme gives the values of the temperature both at the beginning and at the end of the track. Bar = 1.000 pm.

dient becomes stabilized after about two minutes from the open- ing of the water flow inside the capillary tube. Figure 9 reports the track of a creeping organism that is moving not straight toward the capillary tube, but, rather, starting from a substan- tially parallel to the CA-area. The oxytricha creeps at about 130 p d s without any significant variation in velocity with respect to its distance from the capillary tube. At the beginning of the track the creeping oxytricha lies at about 1,800 pm from the capillary tube (14.8' C): the ciliate performs a series of SSR (frequency = 0.23 SSWs) by which it corrects the direction of its movement so reaching the warmer region (CA-area: 16.5" C). Whenever the initial creeping direction of 0. bifaria is clearly oriented towards the capillary tube, the track is uninter- rupted (see Fig. 10, part 111). No looping pattern was ever ob- served under these conditions.

B. 111: A paradigmatic track. The track of an oxytricha mov- ing from about 5 mm (2-area) to the CA-area is given in Fig. 10. In the first part of its trajectory (Fig. 10, part I) the organism performs the typical looping pattern corresponding to a positive variation of temperature of 1.6" C, displacing itself toward the capillary tube. Then the oxytricha performs four SSR (a" = 70 ? 11) before creeping perpendicularly to the thermal gradient

538 J. EUKARYOT. MICROBIOL., VOL. 46, NO. 5 , SEPTEMBER-OCTOBER 1999

Table 1. The ethographic parameters of Oxytricha bifaria in the four different conditions indicated. A = leftward arcs; SSR = Side Stepping Reaction; pSSR = prolonged Side Stepping Reaction; v% = relative percentual frequency, 100% being the sum of the LLE (Long Lasting Elements) and of the SLE (Short Lasting Elements) respectively.

24" C 8.2" C Warm wave front Capillary tube ~ ~~ ~ ~

LLE A Number 195 60 40 46

V % 60.2 55 93 52.1 792 2 325 783 t 309 513 2 247 Radius OLm)

Central angle ( P O ) 77 + 40 73 2 42 113 2 60 62 2 28 Length ( w 0 991 2 516 603 2 348 1,170 2 520 814 2 298

468 2 298 Velocity (P.m/S) 694 2 137 118 f 32 361 t 153

562 2 252

Time (S) 1.4 2 0.6 5.2 f 2.7 3.8 2 2.2 2.2 2 1.5

SLE SSR

pSSR

Number 128 15 - 76 V % 46 16.4 82.6 Correction angle (a") BM: length b m ) 76 t 37 46 t 17 BM: velocity ( P d S ) BM: time (S) 0.16 2 0.08 0.6 2 0.2

-

83 t 28 55 2 22 - 78 2 28 - 91 2 40

478 2 231 73 2 29 - 657 t 370 - 0.16 2 0.06 - - Number - 58

63 V - - - % - - - 175 2 73

- 273 t 171 - - Correction angle (a0) BM: length ( w ) BM: velocity (wm/s) BM: time (S)

BM: radius ( w ) BM: central angle ( P O ) - 120 2 67

- - 158 f 84 -

1.8 2 0.6 - - - - - - 174 f 147 - -

1 AT ("C) 1.6 L (pm) 9,073 t (s) 20.7 D (pin) 911 1, ( p d s ) 438

0.10 Rd ( P d S ) 44

11 0.5 2,348 16.3 2,165 144 0.92 133

I11 2.7 3,148 15 3,05 1 210 0.98 203

IV 1.4 4,085 27.2 1,083 151 0.26 40

(Fig. 10, part 11), experiencing almost no variation of the tem- perature (6T = 0.5" C), because now the thermal gradient has stabilized. A new burst of SSR follows (a" = 70 5 29, n = 5): thereafter, the oxytricha creeps again towards the capillary tube (Fig. 9, part 111; AT = 2.7" C). Once it arrives in the CA-area (Fig. 10, part IV, AT = 1.4" C) it performs a long series of SSR (a" = 76 2 43, n = 18). The value of the I, shown in the upper right panel of Fig. 10 describes the reduction of the velocity following its initial increase; the I, from an initial 0.1 passes to 0.9 and to 0.98 (both describing an almost straight creeping): the Rd describes the general trend of the behavior with its steady increase from part I to I1 and to 111, and its clear drop once CA- area is reached.

C. The behavior in the CA-area. Soon after reaching the CA-area the organisms reduce their velocity to 100 pm/s and start performing several SSR with a temporal frequency of 0.30 & 0.09 (n = 9, see Fig. 7: CA-area). Successively the oxytri- chas move at a higher velocity, keeping themselves inside the CA-area for about 77% of their time (23% out of the CA-area; Fig. 1 l), performing frequent SSR (82.6%: Table 1) at the level of the borderline of the CA-area (Fig. 11). In Table 1, right column, the ethographic data of ciliates in the CA-area (16.5' C) are reported. They creep along A- (52.1%) with the radius measuring 792 pm, their central angle 62", their total length 814 pm, the velocity by which they travel along them being 468 pm/s and their duration in time 2.2 s.

DISCUSSION The physiological potentialities of 0. bifaria, cooled to 9" C

and almost completely inert from the locomotion point of view, were investigated by creating a warm microgradient in a ther- mally isotropic environment: such a warm gradient played the role of a directional stimulus, changing at first both in time and in space (dynamic microgradient), then only in space (static microgradient). Considering that one of the most important bi- ological effects of the cooling process on ciliates is the exces-

BARBANERA ET AL.-THERMAL GRADIENTS AND ADAPTIVE BEHAVIOR 539

1 -area CA - area 1 - area

Fig. 11. An example of a track traveled by an oxytricha in static microgradient conditions in the CA-area and in the contiguous areas, both indicated as “1-area” in the bottom line, being symmetrical with respect to the source of the warm thermal gradient. The temperatures shown in the second line are those measured in the same areas. Bar = 500 p,m.

sively extended period of reversed beating of motor organelles (Inoue and Nakaoka 1990; Machemer 1972, 1974), together with a drop in velocity and increasing inertness (Glaser 1924; Ricci, Barbanera and Erra 1998a), our first result is that oxy- tricha is clearly capable of reacting to the warm wave front by overturning immediately the locomotory status typical of 9” C: (a) the organisms start creeping immediately (b) forwards and (c) at very high velocities. Could such an immediate reaction to the warm stimulus (+ 0.2” CIS) be due to the extra-supply of energy, obviously coming from the heated environment it- self? Ricci, Barbanera and Erra (1998,) reported that the be- havior of oxytrichas isotropically warmed up from 9 to 24” C becomes progressively normal, with a certain, clear delay (s 60 min) from the moment when 24” C are established again: the very prompt recovery (z 1 s) of an active behavior by the oxytrichas kept at 9” C and exposed to the warm wave front, therefore, seems related to the existence of the directional stim- ulus working as an orientating factor (namely as an informa- tional guide for the adaptive behavior) in an otherwise isotropic environment (i.e. without adaptive information). The observed forward movement shown at the arrival of the warm wave front can be explained very well according to several authors (Mach- emer 1989, 1996; Nakaoka, Kurotani and Itoh 1987; Tominaga and Naitoh 1992), who stated that the warming up of the an- terior part of the ciliate hyperpolarizes the cell, inducing, in turn, a forward locomotion.

The second result is that, after recovering efficient locomo- tion, the oxytrichas orient themselves towards the heating source, as perfectly shown by the movement of the centroid of the experimental population (Fig. 5). It moves, indeed, at in- creasingly high velocities as the temperature experienced by the population grows, that is its response rises proportionally to the thermal stimulus. The forward, fast and oriented-to-the-opti- mum creeping represents a clear example of an adaptive be- havior leading the population to the most favorable conditions (Jennings 1906; McFarland 1990; Meyer and Guillot 1990). This adaptive behavior consists of two different components: the thermal-orientation towards the capillary tube and the be- havior keeping the ciliates in the warmest area. The thermal-

orientation expresses the behavioral response to (a) the rising thermal gradient (dynamic microgradient) and (b) the stabilized thermal gradient (static microgradient). The looping patterns represent the most typical response to the arrival of the warm wave front: it is quite constant both in its duration in time and in the number of loops performed (Fig. 7). Moreover they are very similar to the chemosensory behavior of Euplotes vannus creeping in wide circles in the presence of the supernatant of bacteria (Stock, Kriippel and Lueken 1997) although this re- action is performed without any significant variation in velocity. The looping patterns have been interpreted as a sort of “ex- ploratory behavior” in the more general context of the search- ing behavior (Bell 1990). We analyzed the looping patterns by means of the numerical indices and rates already mentioned (Ricci, Barbanera and Erra 1998b). The very constant values of I, (I-area: 0.13; 2-area: 0.12; 3-area: 0.13) strongly support the view that the looping patterns represent a geometrically very standardized initial reaction, during which the “steering wheel” is kept in such a way that the organism does not leave its initial position. The values of I, (I-area: 280 p d s ; 2-area: 353 p d s ; 3-area: 210 p d s ) seem to suggest that the increase of the velocity induced by the warm wave front is smaller as the distance from the heating source increases, possibly due to the reduced velocity of the thermal stimulus itself (Fig. 7: panel 11). However, as regards the more general dynamic effects in- duced by the warm wave front, one must consider the overall patterns reported in Fig. 8: inside the 3-area, indeed, the sudden increase of the instantaneous velocity is clear-cut as in the other two areas, but its general trend shows a weaker physiological response. The finding that the overall displacement of the or- ganisms towards the environmental optimum is very poor (compare the values of R,: 1-area: 38 p d s ; 2-area: 43 p d s ; 3-area: 28 pmls), in spite of their suddenly increased velocity, seems to support strongly Bell’s hypothesis (Bell 1990) that a looping behavior represents an exploratory behavior: the arrival of the warm wave front is the environmental signal triggering the looping pattern, namely a fast environmental scanning.

To evaluate the effects of the warm wave front the ethograms of the experimental populations at 9” C are to be compared (Table 1) with those of organisms performing the looping pat- terns. As far as the LLE are concerned, the oxytrichas creep almost completely along the leftward arcs only of the looping patterns, at very high velocities, while, at 9” C, they perform also linear segments and rightward arcs at very low velocities (Ricci, Barbanera and Erra 1998a). As for the SLE, the pSSR, typical of the cooled populations at 9” C (Ricci, Barbanera and Erra 1998a), as well as the normal type of SSR disappear, com- pletely and instantaneously in the looping patterns. In conclu- sion the looping patterns enable the oxytrichas to explore the environment and to get slightly closer to the heating source by means of both positive (increase in velocity towards the heating source itself,) and negative (decrease in velocity away from the heating source itself) orthokinesis (Diehn et al. 1977; Mach- emer 1996; Van Houten 1979a) as shown in Fig. 8.

When the entire tracks are considered (Fig. lo), the looping pattern (I) is followed by a sort of “behavioral indecision” (11) comprehending a burst of SSR, a straight and slow creeping perpendicularly to the thermal gradient (almost isothermal con- dition) and a final series of other SSR. The end of the looping pattern and the burst of the SSR correspond to the passage from the dynamic to the static microgradient conditions. During the third part of the track (111), leading the ciliate towards the heat- ing source, the oxytricha moves with the highest value of & (203 p d s : compare with those of the parts I, 11, and IV) due both to the very linear creeping pattern resulting from the state of its “steering wheel” (I, = 0.98) and to the propulsion state

540 J. EUKARYOT. MICROBIOL., VOL. 46, NO. 5 , SEPTEMBER-OCTOBER 1999

of its “ciliary engines” (Ik = 210 p d s ) . As soon as the oxy- tricha reaches the CA-area (Fig. 10, part IV), it reduces the velocity (I, = 151 p d s ) and starts performing a continuously interrupted and reoriented track by means of frequent SSR (I, = 0.26): the very small & (40 p d s ) well accounts for the progressive thermoaccumulation of the oxytrichas in the CA- area (Fenchel 1987). In conclusion, while 0. bifaria, as dis- cussed above, reacts to the arising thermal gradient by changing its velocity (looping patterns: positivehegative orthokinesis), under static gradient conditions (Fig. 9), it orientates towards the environmental optimum by repeated SSR, namely by posi- tive klinokinesis (Van Houten 1979a). No significant change of the velocity like those reported by other authors (Matsuoka, Mamiya and Taneda 1990; Tawada and Oosawa 1972) has been ever observed in our experiments.

In the CA-area the repeated SSR (Fig. 11) play two different roles: (a) they interrupt the creeping, thus enhancing the prob- abilities of the oxytrichas of remaining in that favorable area; (b) they enable the oxytrichas to avoid crossing the disconti- nuity between the CA-area and the 1-area, performing the so called “thermal avoidance reaction” (Hennessey and Nelson 1979; Hennessey, Saimi and Kung 1983). These reactions rep- resent a clear example of klinokinesis, closely recalling those already studied for 0. bifaria gathering on smooth surfaces (Ricci et al. 1989), and underneath a floating shelter interfering with the air-water interface (Ricci and Erra 1995). This behav- ior perfectly fits what is known from classic studies also for very complex metazoa, such as the coccinellid larvae (Banks 1957).

The overall picture of the behavior of 0. bifaria under a dynamiclstatic microgradient conditions, beyond revealing a whole series of new behavioral phenomena, perfectly confirmed the primary role played by the changes in temperature, rather than by its absolute values, in determining the motor responses of ciliates (and their adaptive behavior), as discussed from an electrophysiological point of view by Inoue and Nakaoka (1990), and in a far more general perspective by Machemer and Teunis (1996). The findings of Van Houten (1979b), also dis- cussed by Stock, Kriippel and Lueken (1997) about the type I/ type I1 chemoattractants must be considered as a sort of final attempt of interpreting our results from a general unifying point of view. The looping patterns, indeed, might be the expression of electrophysiological changes (general hyperpolarization of the membrane potential, in turn inducing the high forward ve- locity and the disappearing of the SSR) similar to those induced by the type I chemicals. The final accumulation in the CA-area might be similarly due to the thermal conditions, which seem to act quite in the same way as the type I1 chemicals, namely inducing a reduction of the velocity and a contemporaneously increase of the frequency of occurrence of the SSR.

ACKNOWLEDGEMENTS

This paper was supported by grants from MURST and CNR.

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Received 2-25-99: accepted 6-8-99