Electrical Responses of the Carnivorous Ciliate Didinium Nasutum in Relation to Discharge of the Extrusive Organelles

When a hungry carnivorous ciliate Didinium nasutum bumps against a prey, such as Paramecium and Tetrahymena, with the apex of its proboscis, it discharges the extrusive organelles, pexicysts and toxicysts, to capture and paralyse the prey. The captured prey is then drawn towards the proboscis by retracting the discharged organelles into the proboscis. Didinium then engulfs the prey through the proboscis (Balbiani, 1873; Thon, 1905; Jennings, 1906; Mast, 1909; Calkins, 1915; Reukauf, 1930; Dragesco, 1962; Schwartz, 1965). When the specimen collides with inedible ciliates, such as Stylonychia and Euplotes (Mast, 1909), or with the wall of its glass container, it does not discharge the organelles, but instead shows an avoiding reaction due to a transient reversed beating of cilia.

The importance of chemical factors of the prey in the food preference of Didinium has been emphasized for a long time (Seravin & Orlovskaya, 1977; Berger, 1980; Martin & Antipa, 1982). However, the chemical factors of the prey which interact with the proboscis to trigger the discharge of the extrusive organelles still remain unidentified.

Electron microscopical examinations of the proboscis and the extrusive organelles were reported first by Yagiu & Shigenaka (1965). Wessenberg & Antipa (1970) demonstrated that when the organelles were discharged, the toxicysts penetrated into the cell body of Paramecium, while the pexicysts just attached to the cell surface. The mechanism by which the extrusive organelles are discharged still remains unsolved. Recently Hara & Asai (1980) demonstrated that Didinium showed a small hyperpolarization followed by a large depolarization in association with discharge of the extrusive organelles upon contact with Paramecium.

In order to understand the mechanisms by which Didinium recognizes and captures Paramecium, it is important to know the relationship between the electrical events in the membrane and the discharge of the extrusive organelles. We therefore examined the electrical responses of the membrane of Didinium to various kinds of stimuli in relation to discharge of the organelles. Parts of the present works have been reported in preliminary form elsewhere (Hara, Asai & Naitoh; 1980, 1981).

Encysted Didinium nasutum obtained from Carolina Biological Supply Co. (Burlington, North Carolina) were excysted in a wheat-straw infusion. They were then fed on Paramecium caudatum which were cultured in a bacterized wheat-straw infusion. The specimens, which were kept in Paramecium-free culture medium for about 3 h, were washed in a standard saline solution (in mmol I⁻¹: KC1, 1; CaCl₂, 1 and Tris-HC1, 1; pH 7·2) and equilibrated in the solution for more than 5 min prior to experimentation. A single specimen of Didinium was impaled with two microelectrodes and examined by conventional electrophysiological techniques (Naitoh & Eckert, 1972).

As shown in Fig. 1, the anterior end of a Paramecium which was squeezed out through a glass capillary tip (about 30 μm inner diameter) was brought into contact with the apex of the proboscis of the impaled Didinium by driving the capillary toward the Didinium proboscis. The driving velocity was changed from 20 to 250 μm s⁻¹ by changing the rotation velocity of the knob of the micromanipulator.

Discharge of the extrusive organelles was monitored photoelectrically. A phototransistor was placed at the edge of the apex of the proboscis of a magnified (×200) image of the impaled Didinium. The light intensity of the position where the transistor was placed changed when the discharge occurred. All the experiments were performed at room temperature, 20–25°C.

The surface area of Didinium was estimated by assuming that the surface area coincides with that of a rotation body of a certain line. The line was estimated so as to approximate the profile of a photographed Didinium (Fig. 2). The thick line drawn on the x-y plane consists of three portions: the proboscis region, the trunk region and the posterior region. The rotation body is obtained by rotating the line around the y axis.

The membrane potential of Didinium in the standard saline solution measured –40·7 ±3·1 mV (mean and its standard deviation obtained from 20 different measurements). As shown in Fig. 3A (open circles), the membrane was depolarized when

[K⁺]₀ (external K⁺ concentration) was increased in a range from 1 to 16 mmol I⁻¹, while [Ca²⁺]₀ (external Ca²⁺ concentration) was kept constant at 1 mmol I⁻¹. The maximum slope of the potential change was about 30 mV per ten-fold change in [K⁺]₀. The membrane was also depolarized by increasing [Ca²⁺]₀ in a range from 0·016 to 16 mmol 1⁻¹, while [K⁺]₀ was kept constant at 1 mmol 1⁻¹. The maximum slope of the potential change was about 20mV per ten-fold change in [Ca²⁺]₀ (Fig-3B; open circles).

When a weak inward current (less than 0·4 nA, duration 200 ms) was injected into the cell, the membrane potential level shifted exponentially with time as if it were a passive parallel (RC) network (Fig. 4 A,B). The input resistance of a typical cell was calculated from the steady value of the potential shift and the current intensity to be 4·0 × 10⁷ Ω, which corresponds to the membrane resistance of 1·6 × 10⁴ Ωcm². The input capacitance was calculated from the time constant of the passive exponential change (21 ms) to be 5·3 × 10⁻¹⁰ F, which corresponds to a membrane capacitance of 1·3 × 10⁻⁶ F cm⁻². The initial exponential response was followed by a time-dependent decrease in the potential shift, when the current intensity was higher (Fig. 4C).

When an outward current was injected into the cell, the initial passive exponential potential change was followed by an active spike-like depolarizing response (Fig. 4D,F). The peak level and the rate of rise of the response increased with increasing current intensity, approaching their saturated levels. As shown in Fig. 3B (closed circles), the peak level of the spike response shifted towards the depolarizing direction in accordance with increasing [Ca²⁺]₀ in a range from 0·016 to 16 mmol I⁻¹ at a constant [K⁺]₀ (1 mmol I⁻¹). The slope of the shift was about 30 mV per tenfold increase in [Ca²⁺]₀- In contrast, change in [K⁺]₀ (1–16 mmol I⁻¹) at a constant [Ca²⁺]₀ (1 mmol I⁻¹) had little effect on the peak level (Fig. 3A; closed circles). Ciliary reversal was always accompanied by the spike response. More prolonged ciliary reversal was observed when the response was larger.

The spike response was usually followed by a rather slow depolarizing response. The slow response subsided slowly after the outward current was turned off (Fig. 4E,F).

A specimen of Didinium was mechanically stimulated by hitting its surface with the tip (about 2 μm in diameter) of a glass microneedle; which was driven perpendicularly to the cell surface by electrical activation of a piezoelectric transducer upon which the needle was mounted. A weak mechanical stimulus produced a small, slow depolarizing mechanoreceptor potential (Fig. 5A). The amplitude of the receptor potential increased and was eventually followed by a rapid, spike-like depolarizing response when the stimulus intensity was increased. The peak of the rapid response increased with increasing stimulus intensity, approaching its saturation level (Fig. 5B–D).

The depolarizing mechanoreceptor potential was always evoked when a suprathreshold mechanical stimulus was applied anywhere to the cell surface. However the response was smaller when the stimulus was applied to the posterior surface than to the rest of the cell surface (Fig. 6).

As shown in Fig. 7, an impaled Didinium showed a small hyperpolarization when the apex of its proboscis was touched by a captured Paramecium. The approximate time of the touch is shown as a black dot on each potential trace (Vm) in the figure. The hyperpolarization increased as Paramecium was pressed against the proboscis. The rate of the potential change became higher when Paramecium was pressed faster (approximate speed of touch was20μms⁻¹ in A, 50 gms⁻¹ in B and 250 gms⁻¹ in C). The hyperpolarization was not seen when Paramecium was pressed against any part of the cell surface other than the apex.

When the hyperpolarization reached a certain level, Didinium discharged the extrusive organelles, showing a large depolarizing response (Fig. 7). It is noteworthy that Didinium discharged only pexicysts when the contact with Paramecium was very slow and slight (Fig. 7A). Paramecium was not paralysed by bombardment with the pexicysts, but was paralysed by the toxicysts.

Didinium showed a similar hyperpolarizing response and discharge of the organelles upon contact with a nickel-paralysed, non-beating Paramecium (Kuzunicki, 1963) or with an ethanol-deciliated Paramecium (Machemer & Ogura, 1979) as it did upon contact with a normal Paramecium. Didinium scarcely discharged the organelles upon contact with a Triton X-100-extracted (concentration of Triton X-100, 0·01% /vol; extraction time, 30 min at 0°C; Naitoh & Kaneko, 1972) or a heat-killed (1 min at 50°C) Paramecium. The hyperpolarizing response of Didinium to these dead Paramecium, if it occurred, was very small and was followed by incomplete discharge of the pexicysts. Didinium showed neither hyperpolarization nor discharge of the organelles upon contact with the tip of a glass rod similar in size to the anterior end of Paramecium.

Didinium also showed the hyperpolarization and the following discharge of the extrusive organelles upon contact with Tetrahymena, another well known prey item of Didinium, in a manner similar to that shown for Paramecium.

As described in the preceding section, discharge of the extrusive organelles was accompanied by a membrane depolarization. This response consisted of two components, an initial rapid, spike-like component and a long-lasting component.

The time relationship between the discharge and the potential response is clearly demonstrated in Fig. 8, where the potential response (Vm) and the electrical signal corresponding to the discharge of the organelles (D) are displayed together on a CRO screen and recorded. As shown in the figure, the membrane depolarization occurred almost simultaneously with the start of the discharge of the extrusive organelles, which is shown as an upward deflection in trace D. The discharge was never preceded by the depolarization.

The rapid depolarization seen in association with discharge of only pexicysts (Fig. 7A) was identical with that seen in association with a complete discharge of the extrusive organelles (Fig. 7B,C). However, the following long-lasting component of the depolarizing response was different in these two cases. When both pexicysts and toxicysts were discharged, the membrane depolarization lasted for several seconds, showing a small perturbation in the potential level (Fig. 7B,C). When only the pexicysts were discharged, the depolarization subsided within 500–800 ms, followed by repetitive depolarizations. The amplitude and duration of the repetitive depolarizations decreased gradually with time (Fig. 7A).

It is interesting to note that ciliary beating of Didinium had been inactivated during the long-lasting depolarization after complete discharge of the organelles. The beating amplitude was small and the beating direction had been kept slightly reversed. In contrast, conspicuous ciliary reversal took place in association with each depolarization of the repetitive response seen after discharge of pexicysts alone.

As shown in Fig. 9, both the initial spike response and the following long-lasting depolarization were markedly depressed by lowering [Ca²⁺]₀ ( 10⁻⁶–10⁻⁸ mmol I⁻¹ or less) at constant [K⁺]₀ (1 mmol I⁻¹), whereas the preceding hyperpolarizing response was little affected. The initial spike response was followed by a long-lasting hyperpolarization, instead of a depolarization, when [Ca²⁺]₀ was lower than 10⁻⁶ moll⁻¹ (Fig. 9C,D). It should be noted that the discharge of extrusive organelles upon contact with Paramecium was not affected by lowering [Ca²⁺]₀.

When [K⁺]₀ was increased (1–50 mmol I⁻¹), the electrical responses seen upon contact with Paramecium were greatly diminished (Fig. 10). However, the discharge of the extrusive organelles still occurred in high-K⁺ solutions. In fact, the membrane showed a small hyperpolarization instead of a depolarization in association with the discharge of the organelles, when [K⁺]₀ was as high as 50 mmol I⁻¹, hence the resting potential level was slightly positive (Fig. 10F). No initial spike response was observed in such a high-K⁺ solution (Fig. 10E,F).

Neither an inward current, to hyperpolarize the membrane of Didinium beyond the peak level of the initial hyperpolarizing response, nor an outward current, to depolarize the membrane beyond the peak level of the depolarizing response, caused the discharge of the extrusive organelles (Fig. 11A,C). A specimen of Didinium which was depolarized or hyperpolarized by sustained current injection discharged its extrusive organelles upon contact with Paramecium as did the normal specimen. In the depolarized specimen the discharge was associated with a small, spike-like depolarizing response which is indicated by an arrow in Fig. 1 IB. The spike response was followed by a gradual decrease in the level of membrane depolarization. In the hyperpolarized specimen, the discharge was associated with a marked depolarizing spike (Fig. 11D). The membrane had been kept somewhat depolarized after the discharge, even though the cell had been subjected to a sustained inward current.

When the apex of the Didinium proboscis was strongly hit by the tip of a glass needle driven in a direction parallel to the longitudinal axis of the specimen at a speed of more than 4mms⁻¹, which was about 40 times as fast as the normal speed at which Paramecium captured in a capillary were driven against the proboscis, the pexicysts were discharged in association with a depolarizing response without showing the initial hyperpolarizing response. Sometimes the discharge of the pexicysts was followed by a partial, incomplete discharge of the toxicysts.

When a very strong inward current (up to 50 nA) was injected into the cell of Didinium in the standard saline solution, the specimen discharged the extrusive organelles. In contrast, an outward current injection did not produce discharge of the organelles. When the electric charge brought into the cell was relatively small (2·5 ×10⁻¹–4 × 10⁻⁹ C), only pexicysts were discharged (Table 1). The discharge of both pexicysts and toxicysts took place when the electric charge was greater than 10⁻⁸ C. The threshold charge tended to decrease with increasing current intensity. When the current intensity was less than about 15 nA, a sustained injection of inward current did not cause the discharge of the organelles.

When [Ca²⁺]₀ was lowered to less than 10⁻⁸ mol I⁻¹ by adding 5 mmol I⁻¹ EGTA to the external solution, a strong inward current injection did not cause organelle discharge but did produce fusion of the apex membrane. The threshold charge for the membrane fusion was almost identical with that for the discharge of pexicysts in the presence of Ca²⁺. When the intensity of the inward current was increased, the degree of fusion increased and the proboscis moved as though to engulf a Paramecium. It is interesting to note here that discharge of the organelles occurred when 1 mmol 1⁻¹ Ca²⁺ was introduced into the external solution after the apex membrane had been fused by inward current injection. Only pexicysts were discharged when the degree of fusion was small, whereas both pexicysts and toxicysts were discharged when the fusion was large.

The electrophysiological properties of the membrane of Didinium are similar to those of other freshwater ciliates (Naitoh, 1982, 1984). The resting membrane is depolarized by increasing [K⁺]₀ or [Ca²⁺]₀ (Fig. 3). A membrane depolarization by an outward current injection produces a Ca²⁺-dependent depolarizing spike response which is followed by a slow depolarizing response (Fig. 4). The depolarizing response is accompanied by a transient ciliary reversal. These membrane properties of Didinium are consistent with those reported recently by Pape (1982) and Pape & Machemer (1982).

A mechanical stimulation of any portion of the cell body of Didinium produces a depolarizing mechanoreceptor potential which causes a Ca²⁺-dependent depolarizing spike response (Fig. 5). A hyperpolarizing mechanoreceptor potential, such as that seen in the posterior membrane of Paramecium (Naitoh & Eckert, 1969), was not observed in Didinium. The ions which carry the receptor current have not been identified.

The presence of Ca²⁺ ions in the external solution was essential for the discharge of the extrusive organelles by a very strong inward current injection. Part of the current must be carried by the external Ca²⁺ ions, and therefore causes an increase in Ca²⁺ concentration in the proboscis. The discharge was also produced by applying Ca²⁺ ions externally after the apex membrane of the proboscis had been fused by a strong inward current in the absence of Ca²⁺ ions. These results strongly suggest that an increase in Ca²⁺ concentration in the proboscis causes the discharge of the extrusive organelles.

On the other hand, external Ca²⁺ ions were not necessary for the discharge upon contact with Paramecium. Therefore, it is conjectured that contact of Paramecium with the apex membrane causes an increase in Ca²⁺ concentration in the proboscis, probably by the release of Ca²⁺ ions from a hypothetical Ca²⁺ storage site in the vicinity of the extrusive organelles. Yagiu & Shigenaka (1965) reported the presence of a vacuolar system near the extrusive organelles, which is a possible candidate for the Ca²⁺ storage site.

The membrane potential shift by an electric current injection in both the depolarizing and hyperpolarizing direction did not produce discharge of the extrusive organelles, even though the shift exceeded the peak level of the membrane potential responses associated with the Paramecium- induced discharge of the extrusive organelles. Moreover, the depolarizing response was simultaneous with or preceded by discharge of the extrusive organelles (Fig. 8). These facts suggest that the membrane potential responses seen upon contact with Paramecium do not act as triggers for discharge of the extrusive organelles. The initial rapid part of the discharge-associated depolarizing response consists of first - rather slow - and subsequent - faster - components, which are clearly seen as two consecutive peaks in its trace, similar to the depolarizing response to a mechanical stimulation (compare Fig. 7 with Fig. 5). In the depolarizing response to a mechanical stimulus, the first component corresponds to the mechanoreceptor potential and the second component to the Ca²⁺- dependent spike response. These facts strongly suggest that the initial rapid part of the discharge-associated depolarizing response is caused by a mechanical stimulation of the proboscis membrane by the extruding organelles.

The hyperpolarizing response seen upon contact with Paramecium before discharge of the organelles became ambiguous and, in most cases, the discharge did not take place, when Paramecium was extracted in Triton X-100 or killed by heat before the contact. Therefore, the hyperpolarization seems to be correlated with the interaction of the apex membrane with some chemical factors in the Paramecium membrane, which are easily extracted by Triton X-100 or denatured by heat. When the speed of contact of Paramecium with the apex membrane was higher, the rate of the hyperpolarization was higher (Fig. 7). This fact also supports the idea that the hyperpolarization is caused by the interaction of the two membranes.

When the amount of positive charge, hence the amount of Ca²⁺, driven into the cell by a strong current was relatively small, only pexicysts were discharged. Therefore, the Ca²⁺ concentration threshold for the discharge of pexicysts seems to be lower than that for toxicysts. Very slight contact of Paramecium with the apex membrane produced discharge of pexicysts alone. This seems to suggest that the degree of contact of Paramecium with the apex membrane determines the amount of Ca²⁺ released from the hypothetical Ca²⁺ storage site.

Our previous view that Ca²⁺ entry into the proboscis in association with the Ca-dependent depolarizing response causes the discharge of the organelles (Hara & Asai, 1980) is no longer credible.

This work was supported by grants from the Ministry of Education of Japan to Dr H. Asai and Dr Y. Naitoh (411802, 510902, 56105002) and from Mitsubishi Foundation to Dr Y. Naitoh. The first draft of the present paper was prepared during Dr Naitoh’s stay in Ruhr University, Bochum under the sponsorship of Deutsche For-schungsgemeinschaft (SFB 114, TP A5). We thank Professor H. Machemer and Mr C. Pape of Ruhr University, and Dr B. Martinac of the University of Wisconsin for their critical reading of the manuscript.