Properties of Stalk-‘Muscle’ Contractions of Carchesium Sp.

The contractile stalk ‘muscle’ of Peritrichida (Ciliata, Protozoa) has been the subject of several studies. As early as 1912 Koltzoff described the general morphological changes during contraction in Vorticella. Later studies described the anatomy and ultrastructure of the contractile stalk (Favard & Carasso, 1965 ; Randall & Hopkins, 1962; Sotelo & Trujillo-Cenoz, 1959). Propagation of contractility and volume change in contracting stalks of Carchesium have been studied by Sugi (1960, 1961), and biochemical aspects associated with contraction were studied on glycerinated stalks (Amos, 1971; Hoffman-Berling, 1958; Levine, 1956; Townes & Brown, 1965). Contraction cycles of Carchesium and Vorticella were also recorded by cinematographic methods (Jones, Jahn & Fonseca, 1970). Structural and physiological resemblance of the stalk to a metazoan muscle has been stressed in all studies. The small size of the contracting stalk, however, prevented direct measurement and evaluation of its physiological and mechanical properties.

This limitation was partially overcome by a method developed by us (Rahat, Pamas & Nevo, 1969) which enabled attachment to the stalk of Carchesium, and thus made possible measurements of its mechanical properties. This method was based on growing the organisms on glass microscope slides and then attaching small pincers just below the ‘head’. These pincers in turn were attached to a sensitive spring-balance mounted on a micromanipulator.

By this method it was shown that the elastic limit of the stalk was at 1·5 × l0, and the force required for tearing the stalk was in the order of 2−20 kg/cm2.

Further improvement of this method enabled us to record and measure the time course and forces developed by the contracting stalk.

Methods for culture of Carchesium have been described (Rahat et al. 1969). For our present study only specimens with two to five ‘heads’ were used. Experiments were carried out at room temperature of 20−25 °C.

To measure and record directly the forces developed during contraction of the stalk, the following method was used. A microscope glass slide on which a suitable Carchesium was attached was placed in a Lucite chamber containing culture medium (Fig. 1), under a dissecting microscope. A loop of tungsten wire of 20 μm diameter was then tied around the stalk and bent into a hook. This hook was hung on the bent tip of a sensitive metal-wire spring-balance, attached to a micromanipulator. The wire balance was placed between the arms of a U-shaped photoelectric transducer, to be in the path of light source in one arm and a photocell in the other. The responses of the photocell were measured on a 502 A Tektronix oscilloscope and the deflexions of the spring-balance were calibrated up to a weight of 300 μg. Correlation between a DC shift on the oscilloscope and the deflexion of the wire-balance are shown in Fig. 2. The response time of this system was tested using a fast Brush-pen motor and was found to be adequate (less than 8 msec) for our measurements.

A pair of silver electrodes was placed 1−2 mm from the ‘head’ of the stalk. Pulses up to 80 V were required to evoke contraction. In some experiments the stimulating electrodes were attached to the wire spring, and the current was passed directly through the stalk. By this method 5−10 V were sufficient to cause contraction.

The organism responded with a twitch to a single stimulus (Fig. 3). In repeated experiments rise time was 40−60 msec, half-decay time 800−900 msec, and the force developed was between 400 and 800 g/cm2.

Colonies with 2−3 ‘heads’ usually developed forces of 400−500 g/cm2, while colonies with 4−5 ‘heads’ developed forces of 600−800 g/cm2. In older colonies with 15−20 ‘heads’ the main stalk lost its contractility, and the secondary branches only were able to contract. The responses obtained were ‘all or none’ (Fig. 4), the same response being observed at or above the threshold stimulus. Spontaneous contractions sometimes followed contractions evoked by electrical stimuli (Fig. 5) ; in magnitude and duration spontaneous contractions were similar to contractions evoked by electrical stimuli.

The length of the Carchesium attached to the spring-balance was changed by moving the latter with a micromanipulator. At rest length, l₀, normal contractions were obtained (see Rahat et al. 1969 for method of determining l₀). When the stalk was allowed to contract from a length of less than l₀, contractions were not recorded by our system, although they were microscopically visible. No measurements could be made at lengths above l₀, as at such length spontaneous contractions were evoked.

At less than 0·2 stimuli/sec Carchesium contracted at a one-to-one ratio (Fig. 6). At higher stimulation rates of 2 to 14 stimuli/sec, the rate was increased from 0·3 to 1 contraction/sec only (Fig. 7).

No tetanic responses were observed in normal stalks even at 14 stimuli/sec (Fig. 7). In fatigued preparations, however, partial summation, without increase in peak tension, was obtained (Figs. 5, 6). In spite of the ‘all or none’ nature of the twitch partial tensions were developed together with prolonged relaxation time (Fig. 8). Normal contractions were again obtained after several minutes rest.

Electrical stimuli were given by a pair of electrodes placed at various points along the stalk. The point of lowest threshold was always near the ‘head’, and stronger stimuli were required if the electrodes were moved towards the base of the stalk.

To what extent does a protozoan ‘muscle’ resemble a smooth or striated muscle of a metazoon? Is it permissible to define by physiological criteria a contractile element such as the stalk of Carchesium as a muscle?

In a previous study (Rahat et al. 1969) we showed that the tensile strength and elasticity of the Carchesium stalk are similar to those of metazoan muscle. The active mechanical properties of this stalk, however, remained unknown.

In the present study we recorded and measured directly the forces developed by the contracting stalk. The loose connexion of the loop around the ‘neck’ of the Carchesium, and the connexion of this loop to the spring-balance (Fig. 1) certainly affected our measurements. The data presented are therefore minimal estimates of the actual force developed by the contracting stalk. The contractions measured were oxotonic, as the spring-balance was moved by the contracting stalk, while the tension in the latter increased. The twitch of Carchesium is an ‘all or none’ event (Fig. 4). The same conclusion was drawn by Sugi (1961) and by Jones et al. (1970). In fatigued specimen, however, we obtained graded contractions (Fig. 8). Jones et al. (1970) reported that a shortening was completed within 5−10 msec, and Sugi (1960) reported a rise time of 8−10 msec. In our experiments the rise time of tension was 40 msec, and the relaxation time was 1−2 sec.

The results obtained by Sugi (1960, 1961) and by Jones et al. (1970) cannot, however, be compared directly to our observations. In the former cases contractions were isotonic, while in our system the stalk shortened against a load. The movement of the spring-balance reflects the tension and shortening of the stalk. Decrease in contraction velocity of muscle, when the load is increased, is a well known phenomenon, described by P × V = K, where P is the load and V is the contraction velocity (Hill, 1970).

Relaxation time should have been shorter in our system, because of the tension exerted by the spring-balance on the stalk. The slow relaxation of the stalk against the pull of the spring-balance possibly implies a slow decay in the active state of the contracted myoneme. The contribution of the sheath to the slowing of relaxation would be negligible, as the rapid contraction of the stalk was not retarded by it.

Sugi (1961) reported sustained spontaneous contractions, as well as tetanic contractions obtained by repetitive mechanical stimulation. He also suggests that there is a conducting system in the stalk, relaying stimuli to the ‘head’ from which the contractions are initiated. The location, in the cell of Carchesium, of the site at which contractions are initiated is still an open question.

In our experiments the full active state of contraction was obtained by a single twitch, and neither tetanus nor summation was obtained in normal stalks.

During prolonged high-frequency stimulation the stalk contracted and relaxed more or less at its own pace (Fig. 8). This can be explained by refractoriness of a conducting system required to activate the contracting element, or by the inability of the conducting system to exhibit summation.

Amos (1971) recently estimated the minimum work of one contraction of a Vorticella as 6·9 × 10−8 erg, and the instantaneous power during contraction as 2300 calories g−1 h−1. From these estimates the calculated force developed by a contraction is about 20−30 g/cm2. In our experiments on Carchesium a force of 400−800 g/cma was obtained. The discrepancy in the above results can, however, be explained by the different methods at which these forces were obtained. The results for Vorticella were calculated for an isotonic contraction of a stalk against a zero load. According to PV = K (Hill, 1970), however, the forces developed during oxotonic (as in our experiments) or isometric contractions should be higher.

Hoffman-Berling (1958) showed that, unlike what is observed in metazoan muscles, Ca2+ alone is sufficient to produce contraction of glycerinated stalks of Vorticella, while ATP will cause relaxation. Ca2+-induced contractions of glycerinated stalks of Carchesium were also obtained by us (Rahat et al. 1969).

We conclude that while the forces developed by the stalk of Carchesium are similar to those developed by muscles of metazoan cells, its other properties, i.e. summation of mechanical events, tetanic responses and refractoriness, are quite different.

It seems therefore that the contractile system of Carchesium, and probably of other Peritrichida, does not resemble muscles of Metazoa, and nature has produced another efficient contractile system, working probably by a different mechanism.