Protistology 3 (1), 5868 (2003) Protistology The ethology of Protozoa and the “adaptive space” hypothesis: a heuristic approach to the biology of these eukaryotic, unicellular organisms Rosalba Banchetti and Fabrizio Erra* Department of Etology, Ecology and Evolution, University of Pisa, Italy Summary The analysis of the behaviour of the ciliated protozoa led us to explore different fields of their adaptive biology. The neuroethological and the ecoethological studies in our Lab are shortly considered in their aspects of uniqueness (due to the biological peculiarities of protozoa) to support the proposal of a new hypothesis: the “adaptive space” of the protozoa we studied, a unique space defined by their cellular, organismic and environmental parameters. The heuristic value of this hypothesis is then experimentally tested with the study of the case of the double organisms of Oxytricha bifaria, which represent an allomorphic living state of the species. The possible adaptive significance of this allomorphic state is discussed. Key words: behaviour, ciliates, adaptive biology The first and fundamental characteristic of every ‘‘recycled’’ for new adaptive purposes. As an example protozoon is its double nature. It is at the same time a of this process of reuse one could mention the ability perfect eukaryotic cell (functional unit) and a complete of protozoa to change form and functional state (in a organism (adaptive unit). Protozoa, during their long genetically codified and reversible manner), in order existence, have had to face an extremely wide variety to deal, over long periods of time, with different of biological and environmental challenges, and have environmental opportunities or challenges. survived thanks to different adaptive tricks. Such tricks Typical is the case of Oxytricha bifaria, a freshwater are even found in the world of metazoa, even if ciliate, capable of encysting (reorganizing and reducing it own volume to about 1/20 of normal) to avoid extreme conditions of temperature or dryness (Ricci * We would like to dedicate this paper to the memory of Prof. et al., 1985) or producing carnivorous giants (Ricci and Nicola Ricci, who introduced us in the field of ethology of Banchetti, 1993) whenever there is a superabundance ciliates. We’ll remember this exuberant teacher and of potential prey. O. bifaria, by differentiating the giants, researcher for his friendship and his sincere enthusiasm. The radically changes form and function adaptively. The affection and respect, in which he was held, will endure. giants are survival forms of the species when dense © 2003 by Russia, Protistology Protistology · 59 populations undergo severe starvation. They are Continuous Trajectory Change, CTC, Smooth carnivorous predators, namely differentiated states of Trajectory Change, STC, Rough Trajectory Change, the species feeding on new pabula (ciliates instead of RTC, and Side Stepping Reaction, SSR), which bacteria). In this way they reduce the intraspecific mediate the passage from one LLE to the next. competition for the normal bacterial food and from Here we would like to put forward the study of the primary consumers become secondary consumers. The ethology of protozoa as a precious instrument: we will successive steps of the giant formation in O. bifaria are discuss what such a study can contribute to our successive phases of a primigenial cell differentiation, knowledge of protozoa.. that is a reversible phenomenon. This potential tool was the first realization of what metazoa recycled later as 1. THE NEUROETHOLOGICAL APPROACH irreversible cell differentiation. Irreversibility was added only when multicellularity was reached (Ricci and In protozoa the nature of perfect eukaryotic cell Banchetti, 1993). and complete organism coincide: this makes them a The second characteristic is that protozoa are convenient material for neuroethological investi ‘‘small’’, the average dimensions varying from 30 to gations, namely for the study of the relationships 300 µm. This simple ‘‘being small’’ has consequences between behavioural patterns and electrical activities for their biology. (A) As discussed by Purcell (1977), underlying them (Hoyle, 1970; Usherwood and Newth, organisms of these dimensions live at low Reynolds 1975; Ewert, 1976; Kandel, 1976). The very character numbers, namely they experience a peculiar aqueous of neuroethology leads it to concern with the analysis environment, where, no inertia conditioning their of the relations between the manifold organizational locomotion, the viscoelastic forces mainly affect it (we levels that have to interact for the expression of will discuss in another paragraph the consequences of behaviour: from the cellular organelle (for example: the this property of protozoa). (B) All of the environments cell membrane), to the cell (for example: the neuron), are fragmented for them into a myriad of environmental to the tissue (for example: the neural network), to the micropatches, identified by the infinite combinations organ (for example: the ganglion), to the system (for of the minimum variations in space and time of the example: the nervous system), and finally to the various environmental parameters, which support the phenomenon that integrates and expresses all this, local biodiversity of protozoa. (C) Whenever there are namely the behaviour. Huber and Markl (1983) and optimal physiological states and favorable external Cliff (1991) stated that a neuroethological approach conditions, they multiply at extremely high repro iwill be all the more precious the greater is the distance ductive rates in soils, rivers and seas (Finlay et al., 1988; between the interacting levels analyzed. The use of Finlay, 1990). (D) Protozoa, the first eukaryotes to ciliates in neuroethology was proposed (Ricci, 1992) reach the niches of primary and secondary consumer on the basis of three facts: a) the electrophysiology of (Gould et al., 1977) achieved the first ‘‘complete ciliates is a very wellknown field of protozoology ecology’’. These organisms are the successive steps of (Machemer and Teunis, 1996); b) the behaviour is well the microbial food chain, that is the “microbial loop”. studied and understood (Ricci, 1989, 1990); c) the They are large enough to be proper food for both larvae double nature of cell and organism that characterizes and adults of larger metazoa that belong to the protozoa makes possible the direct study of the “macrobial loop” (Azam et al., 1983; Fenchel, 1987). electrophysiology of a single cell (the lowest neuro From Jennings (1906), the first modern scientist ethological level) and its corresponding behavioural to study the ‘‘behaviour of lower organisms’’ with a states (the highest neuroethological level). naturalistic, experimental mentality, the behaviour of Direct attribution of behaviour of freely moving ciliated protozoa has been increasingly seen as an object organisms to spontaneously occurring electrical events of study of great importance for anyone interested in has already been described in Bursaridium difficile (Berg discovering new pieces of the great jigsaw puzzle of and Sand, 1994). In our laboratory an attempt was nature (Ricci, 1981; Ricci et al., 1995; Leonildi et al., made to find in Euplotes vannus spontaneous beha 1998). vioural patterns and then identify their electrical A short preface shoul be given to review the basic equivalents by means of a computerized videotrack elements of an ethogram: all the species studied by us analyzing system to correlate beating of cirri to the creep on the substrate along a piecewise linear path membrane potential of a freely walking organism formed by three Long Lasting Elements, LLE (they (Lueken et al., 1996). It was demonstrated that the last one to several seconds: rightward arcs, A+, creeping velocity of organisms varies spontaneously and segments, S, and leftward arcs, A) and four Short periodically (SlowandFast pattern) in quite a regular Lasting Elements, SLE (they last about 1/10s: manner (T≈0.60.8s) (Fig. 1A,B); the relative memb 60 · Rosalba Banchetti and Fabrizio Erra its manifold cellular or orga nismic meanings by means of neuroethology. 2. THE ECOETHOLOGICAL APPROACH The ecoethological app roach to protozoa may, so to say, “putt us in their shoes”, and lead us to a better understadning of their peculiar environment and equally peculiar environmental biology. Fenchel’s book (1987) had marked the beginning of a real ecology of protozoa, to throw light on the numerous pecu Fig. 1. (A) A tracktype of Euplotes vannus is shown as the succession of the liarities and the importance for positions of the baricentre of the organism in the succession of single photograms the flow of energy and substance of the videorecording; (B) the variation in time of the velocity of the organism that these organisms mediate in from point to point; (C) an analogous pattern of the membrane potential of any environment. The simul this ciliate, spontaneously recurrent in the absence of stimuli. taneous development of etholo gical studies of protozoa, demon rane potential in the same conditions oscillated in a strating the close link between some of their adaptive similar way (T≈0.7s) (Fig.1C). This observation seems characteristics and environmental peculiarities, to suggest that spontaneous bursts of depolarizations indicated that the ecoethological approach was also are responsible for the slowandfast rhythm in freely possible for these organisms (Fig. 2) (Ricci, 1992a, walking cells, by affecting beating of the cirri so that 1996).