THE JOURNAL OF EXPERIMENTAL ZOOLOGY 225337-340 (1983)

RAPID COMMUNICATION Distribution of Photoreceptors Inducing Ciliary Reversal and S w i m m i n g Acce I e r at i o n i n Blepharisma japonicum

TATSUOMI MATSUOKA Zoological Institute, Faculty of Science, Hiroshima University, Hiroshima 730, Japan

ABSTRACT The distribution of photoreceptors was examined with respect to those initiating ciliary reversal and those inducing acceleration of swim- ming. Ciliary reversal was caused when a light stimulus was applied to the anterior end of the animal; the response did not occur when the light was applied to a region within the posterior three-fifths of the animal. On the other hand, acceleration of swimming was induced by light application to both anterior and posterior fragments. These differences in response suggest that the photoreceptors initiating ciliary reversal are different from those inducing acceleration of swimming. Key words Blepharisma, ciliary reversal, photore- ceptor, swimming acceleration

In some ciliates, it has been known that the mechanoreceptors initiating ciliary re- versal are different in distribution from those inducing acceleration of forward swimming. For example, Jennings ('06) found that cili- ary reversal was induced only when the an- terior region of ciliates (Paramecium, Oxy- tricha) was touched with a glass needle, while the same mechanical stimulus to the poste- rior region accelerated forward locomotion. Similar results have been reported in Dilep tus (Doroszewski, '61, '65, '70). Lately, the electric characteristics of the cell membrane during mechanical stimulation have been ex- amined in Paramecium caudatum (Naitoh and Eckert, '69). Mechanical stimulation of the anterior region induces a depolarization of the membrane, while stimulation of the posterior region induces hyperpolarization. Although the cell membrane appears to be differentiated with respect to mechanical stimulation, whether it is similarly differen- tiated with respect to stimulation by visible light remains to be determined. Data of Do- roszewski ('61) on uv stimulation of Dileptus, show that it may be. uv stimulation of the anterior portion (proboscis) induces back- ward movement, but when uv light is applied to the posterior end, Dileptus swims forward.

Work in preparation indicates that Ble- pharisma japonicum shows two kinds of pho- toresponse that cause negative phototaxis. When light is applied to an animal, ciliary reversal is initiated after about 1 second, and

this is followed by acceleration of forward swimming. The present paper reports on the distribution of photoreceptors involved in these responses.

MATERIALS AND METHODS

Blepharisma japonicum was cultured in 0.1% cerophyl infusion containing 1 mM phosphate buffer (pH 7.2) a t 23 k 1°C. The cultures were maintained under periodic light cycles consisting of 12 hours of light and 12 hours of dark. Light intensity was kept a t 10-20 lux. After culture, the animals were concentrated in a glass tube by centrif- ugation. The collected animals were washed twice with a solution containing 1 mM CaC12, 1 mM KC1, and 5 mM TRIS-HCl buffer ad- justed to pH 7.2. The animals were kept in the solution for 2-3 days prior to all experiments.

To examine the localization of light-sensi- tive regions, a glass tube (300 pm in tip dia- meter) connected to a tungsten lamp was used. A microlight spot (3-5 x lo3 lux) could then be applied to definite regions of the an- imal under a dissecting microscope. Light intensity of the background was always kept at lo2 lux. In these experiments, resting ani- mals were used because the light stimulus could be easily applied.

Bisection of the animal was performed with a fine glass needle. Each fragment was kept for 30 minutes a t 5 x 10' lux before every measurement. All measurements using bi-

0022-104X/83/2252-0337$01.50 0 1983 ALAN R. LISS, INC.

338 T. MATSUOKA

sected fragments were completed within 2 hours after bisection because the response was much decreased in fragments kept for a longer time. The number of animals which exhibited backward swimming due to ciliary reversal was counted under a dissecting mi- croscope. Swimming velocity of the frag- ments was measured by using a videotape recorder.

RESULTS AND DISCUSSION

To determine the part of the animal that responded to light stimulation by ciliary re- versal, the microlight spot was applied for periods of 5 seconds each to various regions of the animal or to the whole animal, as shown in Figure 1.

The percentage of the animals showing re- versed ciliary beat, recorded at the right in Figure 1, ranged between 12.3% and 16.3% when the stimulus was applied to the poste- rior one-fifth, two-fifths, or three-fifths of the animal. These responses were probably in- dependent of light stimulation since, on the average, 15.5% of animals that were not stimulated showed spontaneous reversal of ciliary beat.

When the light stimulus was applied to the posterior four-fifths of the animal, the per- centage of animals responding by ciliary re- versal increased to 29.0%, and when the entire animal or only the anterior one-fifth of the animal were irradiated, almost all of the animals responded by reversed ciliary beat and backward swimming. In this exper- iment the intensity of the light stimulus was maintained at 3-5 x lo3 lux. The apparatus did not permit localization of the stimulus at higher light intensities, so that it was impos- sible to determine whether the refractoriness of the posterior end might be due to a much higher threshold than that of the anterior end.

But bisected animals (see Materials and Methods) were viable, responded to light, and could be stimulated in toto by much higher intensities. As in the previous experiments, light stimuli, ranging in intensity from 5 x lo3 to lo5 lux, were applied to the fragments for 5 seconds and the percentage of individu- als responding by ciliary reversal and back- ward swimming was then determined. Figure 2 shows that almost 100% of anterior frag- ments responded to stimulation at every light intensity. In the posterior fragments, by con- trast, the degree of response did not change in spite of a 20-fold increase in light inten-

p o r t i o n o f l i g h t r e a c t i n g c e l I s ( % 1 a p p l i c a t i o n

14.0 f 2.6

16.3 f 4 . 5

95.7 f 2.1 a -

Fig. 1. Rate of the ciliary reversal induced by partial light stimulation to the cell. Light was applied to the blank portion of the diagrams. Figures written at the right of diagrams show the means of five identical mea- surements (lo2 cells per measurement) and S.D.S.

sity. The regression line for the posterior fragments corresponds to 20%, a value which agrees with the spontaneous response level of 19%. It is thus clear that application of light to the posterior part of the animal can- not induce ciliary reversal, due to the fact that photoreceptors that initiate ciliary re- versal probably do not exist in the posterior part of the animal.

Nevertheless, the ciliary system, including depolarization-dependent Ca2+ channels, is apparently still functional because spon- taneous ciliary reversal can still occur and because ionic stimulation causes ciliary rever- sal in posterior fragments as well as in ante- rior fragments.

When the intensity of the light to which Blepharisma are exposed is increased above the background level of lo2 lux, the rate of

DISTRIBUTION OF PHOTORECEPTORS IN BLEPHARISMA 339

100

h

M Y

f 50 0

CL v) D)

L

-

-

I I l l

1 5x103 lo4 2 4 6 810'

I u x

Fig. 2. Effect of high light intensities on ciliary rever- sal in the anterior (open circles) and posterior fragments (solid circles). Abscissa indicates light intensity (lux) in a log scale. Points and attached bars correspond to the means of four identical measurements (30 cells per mea- surement) and S.D.S.

forward swimming is accelerated and reaches maximum velocity after about 5 minutes of illumination (work in preparation). It was impossible to keep a microspot of light fo- cused on a part of the animal during such a long period of time; hence, determining re- gional distribution of photoreceptors on whole animals proved to be impossible. But this difficulty could be circumvented by the use of anterior and posterior fragments ob- tained by bisecting the animal. These frag- ments could then be illuminated in toto at various light intensities. In the experiments summarized in Figure 3, swimming velocity was measured after light had been applied for 10 minutes. At 5 x lo2 lux, the mean velocities of the anterior and posterior halves were 109 pdsecond and 92 pdsecond, re- spectively. In both fragments, an abrupt in- crease of swimming velocity was observed from 5 x lo2 to 3 x lo3 lux; further increases in intensity were without effect. At 3 x lo3 lux, the swimming velocity was 249 pdsec- ond in the anterior half and 287 pdsecond in the posterior half. Thus, the swimming velocity was found to be strikingly acceler- ated in both fragments. These results imply that the photoreceptors inducing the acceler- ation of swimming exist on both anterior and posterior regions. The present results sug- gest that the distribution of photoreceptors

300 ~ - u u n

\ -4- - 5 200 - -+- " 0

0 ' I

0 1 0 ' 2 3 4 5 10'

I U I

Fig. 3. Effect of light intensities on swimming veloc- ity in the anterior (open circles) and posterior fragments (solid circles). Points and bars correspond to the means and S.E.S. (determinations of 10' cells).

initiating ciliary reversal is different from that of the receptors inducing acceleration of swimming. The former appear to be located in or on the anterior end of the animal, while the latter are possibly distributed over the entire cell.

In another ciliate, Dileptus, mechanical stimulation of the anterior half induces cili- ary reversal, while stimulation of the poste- rior half starts forward swimming (Do- roszewski, '70). In Paramecium, a density gradierk of mechanoreceptors whose stimu- lation results in ciliary reversal exists from the anterior toward the posterior end, while the gradient of mechanoreceptors leading to acceleration of forward locomotion occurs from the posterior to the anterior end. (Ogura and Machemer, '79). In the photoresponse of Blepharisma, the photoreceptors initiating ciliary reversal are localized on the anterior end, while those accelerating forward loco- motion probably exist over the whole region of the cell. Thus, the distribution of photore- ceptors on B. japonicum appears to be differ- ent from that of mechanoreceptors in other ciliates. Naitoh and Eckert ('69) examined the potential changes resulting from me- chanical stimulation in Paramecium. Me- chanical stimulation of the anterior portion caused a transient depolarization of the membrane, while stimulation of the poste- rior portion induced hyperpolarization. De- polarization of the membrane produced an increase in permeability of the membrane to Ca2+ ions and thus an intlux of Ca2+ ions (Eckert, '72). In Blepharisma, ciliary reversal caused by light stimulation may also be de- pendent upon the local depolarization of the

340 T. MATSUOKA

anterior membrane which would be closely followed by Ca2+ influx. In contrast to ciliary reversal, acceleration of swimming due to light stimulation is probably performed mainly by an increase of beat frequency of cilia. The hyperpolarization, which is caused by an increase of permeability of the mem- brane to K+ ions (Naitoh and Eckert, '73), is accompanied by an increase in frequency of ciliary beat (Kinosita et al., '64). The results of these studies suggest that light-induced acceleration of swimming in B. japonicum is presumably achieved by an increase of beat frequency and that K+ ions may be involved. The orientation of cilia in glycerol-extracted Paramecium is toward the posterior end in Ca2+-free solution, but the orientation changes toward the anterior end when Ca2+ concentration is increased (Naitoh, '69). In Blepharisma, swimming velocity may be al- tered by an angular change of ciliary beat which is dependent upon intracellular Ca2 +' concentration. Photoreceptors, which induce acceleration of swimming, possibly lead both to an increase of beat frequency and to the orientation of cilia toward the posterior end.

The present paper suggests that different kinds of photoreceptor in Blepharisma lead to two kinds of photoresponse. Further work

will involve determining, by means of action spectra, the characteristics of the two kinds of photoreceptors and also the transmission process between receptors and effectors.

LITERATURE CITED

Doroszewski, M. (1961) Reception areas and polarization of ciliary movement in ciliate Dileptus. Acta Biol. Exp., 21:15-34.

Doroszewski, M. (1965) The response of Dileptus cygnus to the bisection. Acta Protozool., 3:175-182.

Doroszewski, M. (1970) Responses of the ciliate Dileptus to mechanical stimuli. Acta Protozool., 7:353-362.

Eckert, R. (1972) Bioelectric control of ciliary activity. Science, 176:473-481.

Jennings, H.S. (1906) Behavior of the Lower Organisms. Columbia University Press, New York.

Kinosita, H., S. Dryl, and Y . Naitoh (1964) Relation between the magnitude of membrane potential and ciliary activity in Paramecium J. Fac. Sci. Univ. To- kyo Sect. IV, 10:303-309.

Naitoh, Y. (1969) Control of the orientation of cilia by adenosinetriphosphate, calcium and zinc in glycerol- extracted Paramecium caudatum. J. Gen. Physiol., 53:517-529.

Naitoh, Y., and R. Eckert (1969) Ionic mechanisms con- trolling behavioral responses of Paramecium to me- chanical stimulation. Science, 164:963-965.

Naitoh, Y., and R. Eckert (1973) Sensory mechanism in Paramecium, 11. Ionic basis of the hyperpolarizing me- chanoreceptor potential. J. Exp. Biol., 59:53-65.

Ogura, A,, and H. Machemer (1979) Topographical differ- ence of mechanoreceptor currents in Paramecium cau- d a t u m Zool. Mag., 88:529.