The crowding effect: an ethologic analysis

Europ. ]. Protisto!' 31, 302-308 (1995) August 25, 1995

European Journal of

PROTISTOLOGY

The Crowding Effect: an Ethologic Analysis Nicola Ricci and Fabrizio Erra Dipartimento atScienze dell'Ambiente e del Territorio, via A. Volta 6, Piss, Italy

SUMMARY The phenomenon of crowding of ciliates in rest areas has been described previously [23]: these organisms tend to collect on the bottom of an experimental apparatus under objects interfering with the water-air interface. The behaviour of Oxytricha bifaria has been analyzed as a basic element possibly accounting for the effect itself: to monitor the phenomenon properly a well-defined time (1 h 15 min) was chosen during the formation of the overcrowded population; in addition, well-defined areas were TV recorded, the populations within them studied and their behaviour analyzed and compared. The results obtained are the following: (a) the number of idle cells is significantly larger in the Shelter-Area (S) than in the Open (0); (b) there is a net inward flow of oxytrichas, constantly enhancing the cell density in S; (c) the ethogram of oxytrichas creeping into S is quite similar to that of the populations in 0: only the creeping velocity in S is smaller than in 0; (d) the frequency of Side Stepping Reactions performed by O. bifaria at the level of the border between Sand 0 is 5 - 6 times higher for oxytrichas creeping outwards than for those entering S. The combined effect of these phenomena well accounts for the formation of the crowding conditions.

Introduction A new phenomenon, the crowding effect, has been recently described [23]: when a droplet of physiological medium is put on a microscopic slide and a small piece of coverslip (kept still by an anchored glass arm) lies at the level of its upper surface, the hypotrichs creeping on the bottom progressively gather under the coverslip itself into the so-called "rest-area". The effect was found to depend upon the microvibrations of the water-air interface and its sigmoidal temporal formation was described. This crowding effect seems to play important adaptive roles in the biology of the species, enhancing its probabilities of finding the proper microenvironment [15, 22] where to feed, to mate, to encyst. We tried to deepen our understanding of both the phenomenon itself and its relevance for the species: we

The research was supported by grants from M.U.R.S.T. and C.N.R. 0932-4739-95-0031-0302$3.50-0

analyzed the motility of a population of O. bifaria, exposed to the proper experimental conditions [1]. As stated by Jennings in 1906 [6] and very recently by Fenchel [3] and by Ricci [14, 16, 18], the behaviour of any ciliate must be viewed not simply just as its "cell motility" (cyclosis, mitosis, micronuclear migrations etc. do not play any role in interfacing the organism with its environment), but, rather, fully as the "adaptive behaviour" of the species, thus meaning the entire range of behavioural patterns used by the same species to keep itself within the survival limits of its own biology, according to the definition given by Meyer and Guillot [10]. The adaptive behaviour represents the fast way for a species to give the best answer to the risks of its daily life, namely to adapt rapidly to both its physiological needs (feeding, reproducing, mating... ) and to the environmental challenges (exploring, escaping, dispersing... ) [24]. An accurate analysis of the behaviour of a species (namely of as many different patterns and parameters as possible), therefore, cannot but help us to penetrate its adaptive strategies (as clearly suggested by Whitman [25], by Heinroth [5], by Lorenz [7], and more recently © 1995 by Gustav Fischer Verlag, Stuttgart

Crowding Effect in Oxytricha . 303 by Martin and Bateson [9]): as a consequence, it becomes possible to try to understand the "crowding" phenomenon just as O. bifaria experiences it. The study of the behaviour of experimental populations of O. bifaria under crowding-inducing conditions was undertaken on the basis of both (a) the theoretical framework, discussed by Ricci [16], regarding the possibility of analysing both qualitatively and quantitatively the creeping behaviour of ciliates (namely their ethogram, sensu Eibl-Eibesfeldt [2]) and (b) the successful attempt at synthesis, namely of "putting the pieces together" again [12]. Material and Methods 0. bifaria is a freshwater hypotrich: the strain LV06 was used throughout the experiments described here. The standard culturing protocol [19] was followed: the cells, collected by a mild centrifugation at about 100 g for 5', were tested six hours later, to ensure their complete recovery from any possible acceleration stress. To study the phenomenon and the behaviour of 0. bifaria, the standard "observation apparatus" described by Ricci [17] was set up (Fig. 1 Aj, A2): the

fluid (0.5-1 ml in volume) was the uninoculated lettuce medium [19]. As shown in Fig. 1 B, six rectangular areas (0.77 x 1.54 mm) of the bottom of the standard apparatus were individuated on the TV screen and studied specifically for the behaviour of the oxytrichas: the first 3 lay under the fragment of coverslip and have been called Sj, S2' and S3'respectively, the first laying right along the edge of the coverslip (H-line), the second being contiguous to the first, the third, separated from the first two, corresponding to the innermost area of the "shelter"; the other three stripes were covered by the water-air-interface and called OJ, 02' 03, respectively, each being symmetrical to the corresponding one among the first three. The TV recording sessions in dark-field conditions followed the protocol described by Ricci [17]. The experiments were run at a temperature of 21 ± 1 "C. The video tapes were then scored frame by frame to study (a) the successive positions of the bodies of the organisms, (b) the idle vs the mobile oxytrichas, (c)the flows of the organisms in the different directions, (d) their ethograms. The terminology we use in this paper is described thoroughly by Ricci [16]. The four behavioural patterns mediating the clockwise changes of the creeping direction of 0. bifaria (the so-called Short-Lasting Elements, SLE) are: the Continuous Trajectory Change (CTC), the Smooth Trajectory Change (STC),the Rough Trajectory Change (RTC), the Side Stepping Reaction (SSR). Their effects on the geometry of the tracks are progressively stronger from the first to the fourth, both for the creeping velocity (unchanged for CTC, strongly reduced for STC, reduced to zero for RTC, negative during the backward motion of the SSR) and for the correction angle (CTC: ~ 8°; STC: ~ 22°; RTC: ~ 35°; SSR: ~ 63°). The data we obtained were then tested by the Kruskal-Wallis non-parametric test (H-test).

PLA STI CI:-l E

Results

1. The Spatial Distribution of the Populations

B o

0,

0,

0,

S,

s.

S,

.22l.m.m.1 19

moo 1 1'

MI\lI

COVER SL I P

Fig. 1. The standard observation apparatus. Aj. General scheme; A2. Schematic side view to show how the coverslip relates to the water-air-interface. B. The areas studied experimentally and their relationship with the entire piece of coverslip; H-line: the ideal line which divides the areas under the shelter (Sj) from those in the open (OJ).

In our conditions, the steady state of the crowding was obtained 1 h 35 min after the onset of the experiment: we chose to study the behaviour of experimental populations after 1 h 15 min in order to avoid the chaotic phenomena and the lack of any clear cell flow at the very beginning of the observation. Each figure of Fig. 2 is the average value of 7 observations, each made at 0, 10,20,30,40,50 and 60 min, to scan that particular minute (between 1 h 15 min and 1 h 16 min) we had decided to study. The results shown in Fig. 2 demonstrate that: (a) the largest difference between 2 areas, as far as the number of cells is concerned, occurs between 53 and 0 3 the first being roughly ten times as large as the second; (b) while the control-area (03) is almost empty of cells, this number steadily increases to 02, 0 10 5 10 52 and 53' the largest difference being that between 52 and 53 (.1cells = 16 cells!); (c) all the differences are highly significant (p < 0.01); only between the 0 1 and 51 areas, p is a little higher (p = 0.02). 2. The Flow of Oxytrichas in Oland S1 Areas Two areas, 0 1 and 5 10 were studied to measure the flow of oxytrichas, because they are contiguous, being

304 . N. Ricci and F. Erra I(

line

H line

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W A I

c o v ers lip

35

29.4

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Fig. 2. The schematic drawing shows the number of cells measured in the different areas: the char acte rs indicate the mean; the standard deviation is reported as a vertical bar; for all the areas the number of the observations is n = 7; WAI = water air interface.

fig . 3. An example of our way of studying the inward/outward flows of the cells: each cell drawn with a solid line represents its initial position (either in 0 1 or 51' respectively); each cell dra wn with a dott ed line represents the final position (after 1"); the arrows indicate the movement; when the initial (or the final) positions were externa l to both 0 1 and 51> they were not drawn. The shadowe d oxytric has represent idle organisms.

divided by the H-line (the line corresponding to the edge of the coverslip) , and have the most similar cell density. The sample minute we had chosen was monitored by 13 observations (1 sec every 5 sec) at 0, 5, ... , 55 and 60 min, respectively: on an acetate sheet put directly on the TV screen the initi al and the final positions

of all the cells present in O land in S1 were outlined; an example of what was obtained for each observation is given in Fig. 3. 2. A. Creeping vs Idle Oxytrichas. In 0 1 3.1 ± 1.7 creeping and 2.2 ± 1.1 still oxytrichas were observed on avera ge (n = 13), while in 51 the corresponding val-

Fig. 4. The average values (bold figures ± standard deviat ion; n = 13 for all the observations) of the moving cells through the different sides of 0 1 and 51 in the different directions. Th e thick black line indicat es the H-line. To make a comparison betwee n the total s, the number of cells creeping in north-south direction must be multiplied by two (4.8 x 2), becau se the northern and southern sides of the rectangle are half of the eastern and western sides.

01 TOWARDS

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S E W

81

INWARDS

OUTWARDS

~ ~

0

-S

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~

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O.6±O.7

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OUTWARDS

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TOTAL FLOW NORTH

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SOUTH

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O.7±O.9

4.8 EAST

Z.1±1.6

ty.Z.8±1.7

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WEST l.2±1

13.3

Crowding Effect in Oxytricha . 305

cells/sec) than that of those moving north-south (4.8 x 2 = 9.6) (p < 0.01); (b) the east-west flows are unbalanced: as shown in Fig. 5, 1.5 oxytrichas/ sec pass from O 2 to 01> 2.1 pass to 51 (through the H line) and 2.8 leave 51 for 52; 1.4 westward cells leave 52to pass to 51,1.2 cells cross the H line to 01> and only 1 westward cell moves forward to 02: the three partial balances are of + 0.5, + 0.9 and + 1.4 cells, respectively, the + indicating movement in eastward direction. Therefore we conclude that the eastward flow (6.4 cells/sec) is larger than the westward one (3.6 cells/sec); while the first increases eastwards, the second decreases progressively westwards: the result is a measure of constant accumulation of the cells in the 5 areas. These results seem to indicate that (a) the cells are trapped in the 5 areas; (b) the crowding effect is also seen in 0 1-02 areas (as already shown by the cell densities).

COVERSLIp I

areas

O2

cell densities eastward flow westward flow balance of the flows Fig. 5. A schematic representation of the different areas studied (02, 01' 51> 52), of their cell densities (x ± s; n = 7), of the eastward and of the westward flows, and of the balances of the contrary flows: the figures in the arrows indicate the number of cells/sec passing through the borders shown in the scheme: the eastward and westward flows have the same values as in Fig. 4.

ues are 3.2 ± 1.5 and 3.5 ± 1.4, respectively; thus, while the moving organisms are the same in 1 and 51> the still cells are far more frequent in 51 (p = 0.015). 2. B. The Direction of the Flows. In 0 3 all the oxytrichas creep without any preferential direction; in 53 the study is hindered by the overcrowded conditions. For both 0 1 and 51 we measured the number of oxytrichas either entering or getting out through each side of the rectangle (Fig. 4). It must be recalled that the rectangles are 0.77 mm wide and 1.54 mm long: the east-west flow therefore must be expected to be twice as much as the north-south flow. Several conclusions can be drawn from the data shown in Fig. 4: (a) the number of cells moving in the east-west direction is larger (13.3

°

3. The Ethogram of Oxytricha in 0

1

and 51

Study of the flows showed that the creeping behaviour toward the 5 areas accounts for the crowding-effect. On the basis of the same rationals already discussed for the analysis of the flows, two ethograms were drawn: (a) for oxytrichas creeping in 0 1 and (b) for those creeping in 51' No significant difference exists between the 0 1 and the 51 ethogram as far as the Long Lasting Elements (leftward arcs, A-, and segments, 5) and their parameters (radius, central angle, length and duration) are concerned. Only the velocities along Aand 5 are lower in 51:A-: 0 1256 ± 71l-un/sec, n = 37, 51 211 ± 43 urn/sec, n = 43, p = 0.031, 5: 0 1297 ± 118 urn/sec, n = 36, 51 245 ± 104, n = 53, P = 0.039. This finding could be expected on the basis of many idle cells in the 5 areas (zero velocity). As to the Short Lasting Elements, neither the frequencies nor the correction angles (IX) of CTC, 5TC, RTC are affected. The Side Stepping Reactions (55R), on the contrary, represent the most relevant behavioural element, as far as

Table 1. The numbers of 55R per minute in the different areas are indicated in the top line. For each value the mean and the standard deviation (bold characters) are indicated: n = 5 for all the observations. In each of the 8 central boxes (OJ, 51), the ratios between the average number of the different kinds of 55R and the total number of mobile oxytrichas is indicated by italic characters AREA5 55R observed:

03

01

51

53

27 ± 3.7

56.3 ± 3.6 19.3

75.4 ± 3.2 23.5

99.8 ± 3.3

spontaneous

17.4±3.7

17.3 ± 1.8 5.6

19.6 ± 1.1 6.1

7.2 ± 1.6

upon-bump

9.6 ± 3.8

39 ± 4.4 12.6

57.8 ± 5.4 17.4

3.4 ± 1.1 1.1

18 ± 5.3 5.6

total n°

at the H-line level

92.6 ± 5

306 . N. Ricci and F. Erra

the crowding phenomenon is concerned. As shown in Table 1 the total number of SSR per area increased from 27 (in 03) to 56 (in 01) to 75 (in Sl) to 100 (in S3)' Each of these values, in turn, results from the spontaneous SSR (17 in 03' 17 in 01> 20 in S1> 7 in S3) and the SSR performed upon cell-to-cell bumps (10 in 03' 39 in 01> 58 in S1> 93 in S3)' The overall result is that the spontaneous SSR are more or less the same in 03' 0 1 and S1> whereas in S3 they are almost completely "masked" by those elicited by the very frequent bumps from overcrowding. When the ratios between the number of the SSR observed and the number of mobile cells are made for the 0 1 and Sl areas, the differences become far smaller: while in 1 the total number of SSR is 19 per mobile cell (= 6 spontaneous SSR + 13 upon-bump-SSR), in Sl it is 23 (= 7 spontaneous SSR + 16 upon-bump-SSR). The conclusion, based on these data, is the following: in the S areas the SSR frequency is affected significantly (p = 0.046) by the crowding effect. A clear result refers to the SSR performed at the level of the H-line by cells which avoid passing from 0 1 to Sl and vice versa (Table 1, fourth line). In this case, the difference is highly significant (p = 0.004) with the ratio (SSRlmobile cell) 1.1 for 0 1 and 5.6 for Sl' This result demonstrates that (a) the H-line is actually perceived as a spatial discontinuity, (b) the ciliates avoid escaping from the Sl area 5 - 6 times more frequently than entering it, and (c) also the cells creeping toward the S area perceive the environmental discontinuity (between the and the S areas) and avoid it, although far more rarely than the others.

°

°

Discussion The results here reported confirm that the study of behaviour helps to understand the crowding effect. The data we report in this paper demonstrate that the 10-fold higher cell density in S3 is due to several factors working synergistically: (a) the "inward" flow of oxytrichas, (b) the inertness of the cells lying in S3 and (c) the number of SSR performed when an oxytricha creeping outwards manoeuvers in correspondence with the H-line (d. Fig. 5 and Table 1). The data we obtained, however, deserve a far deeper consideration, since they are capable of revealing a series of important co-factors acting either in cooperation or in contrast with those mentioned above. The first consideration is that the crowding of oxytrichas in the Rest Area (namely the occurrence of the "Shelter Effect") is the expression of two counteracting tendencies: the first leads the cells to gather underneath the coverslip through the three mechanisms already cited, while the second makes the overcrowded oxytrichas spread outwards, namely from the S areas to the areas, as demonstrated by the dispersal kinetics of the population in the Rest Area, as soon as the coverslip is taken away [23]. According to this point of view, the

°

steady-state is reached when the "attraction" towards the Rest Area is exactly counterbalanced by the "repulsion" induced by the overstimulation occurring among overcrowded cells. It must be clearly stated that as "attraction", we mainly refer to the effects of the inertness and the stillness induced by the floating coverslip: these effects are expressed both by the large number of still cells and by the reduced speed of the creeping ones. How to account for these findings? It has been observed in E. crassus (Camodeca N., personal communication), that resting cells, in the absence of external stimulation have a membrane potential of - 30, - 35 mV (instead of the normal - 25 mV) and that, under these conditions, the ciliary organelles are immobile: if the same should also be found to be true for O. bifaria, we could hypothesize that, under the coverslip, that is to say, far from the continuous "rain" of microvibrations produced by the water-air-interface [23], its basic membrane potential drops from normal values, typical of alert conditions, to lower values characterizing a cell with generally depressed activity (d. also [8]). This consideration must be made, because the idle cells are somehow "unexpected", in normal populations of O. bifaria [13]: under the shelter the idle oxytrichas are just "resting" organisms without the normal "awaking" stimulations which make them move continuously. Provided that the electrophysiological interpretation of the phenomenon may prove to be correct and that nothing is known about the nature of the specific receptors for the microstimulations that can affect the general state of the membrane potential it may be assumed that perhaps the dorsal bristles are involved in this phenomenon, as they are supposed to be in rheoception [4]. The SSR is performed spontaneously and typically used by ciliates to interrupt their subcircular tracks [11], only more rarely is it induced by the environment (different stimulations): in the latter case it assumes the peculiar significance of a true Avoiding Reaction, as described by Jennings in 1906 [6]. This is the case of the SSR performed by O. bifaria creeping from S, to 0 1 in correspondence with the H-line: here the SSR actually are behavioural responses capable of keeping the cells under the shelter, in much the same way as they proved to keep the oxytrichas on the smooth surface whenever they came in contact with the rough substrate [21]. A further consideration about the short-lasting elements of the ethogram [14] and their adaptive significance is to be made with regard to the relative frequencies of CTC, STC, RTC and SSR: as stated in the Results section, the coverslip does not affect anyone of them, but it affects the SSR! Such an observation finds strong support in the findings already reported in literature about the behaviour of populations of Euplotes crassus [20] and the behaviour of O. bifaria moving on rough surfaces [21]: only the SSR frequency is affected in both cases. It must be said that, although we measured the SSR occurring at the level of the H-line, this line is perceived

Crowding Effect in Oxytricha . 307

Refer ences

2 3 4

5 6 H line

Fig. 6. Th e general scheme of the "C rowding Effect", as visualized on the basis of the cell densities and behaviour al peculiarities in the different areas . The vertica l part of the slope (in cor respondence with the H-line) has been introdu ced to show how difficult it is for an O. bifaria to leave the S1 area under the pressure of the crowding-effect (track B).

by oxytrichas ra ther as a gra ded discontinu ity. An oxy tricha creeping in th e 51 area tow ards th e 0 1 has an increasing probab ility of performing an 55R up to the H -line, beyond which suc h a probab ility starts dropping aga in down to zero. T hese results seem to be in reasonabl e agreeme nt wit h the microvi brationsfield hypothesis [23]: the substrate corresponding to th e 0 2, OJ, s., 52 are as shou ld be exposed to progressively sma ller intensities of perturbat ions, thus creating a gradient from the outside to th e 5 areas capable of lead ing the oxytrichas to the most protected area (53)' Th e sketch, drawn in Fig. 6, schema tically shows a way of visualizing the crowd ing effect: a sort of "depr ession " , whose depth, by an d large, is pr oportion al to the cell densities in the different areas we stu died. The general steepness of these curves mirro rs the "attract ion " exerted by the shelter area itself on the cells cas ually creeping in 52 an d in 51' T he critical step at the level of th e H -line has been drawn to indicate th e "obstacle" clearl y perceived by th e cells creeping outwards from 51 (under the crowding press ure) and stro ngly avoi ded by them by performing freq uent SSR.

7 8

9 10

11 12 13 14 15 16 17

18 19

Acknowledgements The authors wish to thank Dr. R. Banchett i not only for her editorial skill and patience, but also for her helpful suggestio ns and criticisms.

20 21

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Key words: Ciliates - Shelter effect - Ethogram - Adaptive beha viour Nicola Ricci, Dipartimento di Scienze dell'Ambiente e del Territorio, via A. Volta, 4,56100 Pisa, Italy