THE FUNCTION OF THF EYESPOT IN CHLAM YDOMONAS

[ 292 ] THE FUNCTION OF THF EYESPOT IN CHLAM YDOMONAS BY J. N. Department of Botany, University of Manchester {Received 7 Jamiary 1953) (With I figure in the text) INTRODUCTION The function of the eyespot in flagellates in general, and in Chlamydomonas in particular, has been the subject of much discussion, but a definite conclusion which is generally acceptable has yet to be reached. This indecision has been mainly due to the inability to make any truly direct approach to the problem. Until methods in microtechnique become sufficiently refined to allow the removal of eyespots from individual cells, it appears inevitable that discussion of the subject must continue to be based on indirect evidence only. By its very name which has long been in common usage, the eyespot is assumed to be photosensitive, and its similarity in colour to the true eyes of certain lower animals established this assumption long before there was any experimental evidence. Strasburger (1878) was among the first to bring a more critical attitude towards the subject by pointing out that many flagellates without eyespots were nevertheless reactive to light. He postulated that sensitivity to light was a property of the entire protoplasm in those organisms which showed it. Soon afterwards Engelmann (1882) published the results of experiments with Euglena. In this organism the eyespot is situated at the anterior end of the cell. He passed a sharply defined shadow forward along the length of a cell swimming towards a source of light, and observed that this had no effect until the shadow covered the region of the eyespot. At this point the cell shortened itself and turned away from the original direction of motion. Mast (1927) claimed to have repeated and confirmed this experiment. Luntz (1931), however, was unable to produce a shadow with an edge so distinct that part of a cell could be shaded and the rest in the light, and he questioned the validity of Engelmann's results. Such criticism may be unjustified, but the value of discussing it in this paper is doubtful since there is good reason to believe that the eyespots of Euglenophyta on the one hand, and of Chlorophyta on the other, are structurally and functionally dissimilar. In Euglenophyta the eyespot consists of an irregular mass of pigment granules, whereas in Chlorophyta the eyespot has a definite outline and appears to be more homogeneous. A complex structure, including a cup-shaped pigment layer and a lens which concentrates the light, has been postulated for certain members of the Volvocales (Mast, 1927). In addition to these structural differences, the position of the eyespot is significant. In Euglenophyta the eyespot is always located at the anterior end of the cell close to the base of the flagellum. Wager (1900) observed in Eitglena an enlargement of the base of the flagellum adjacent to the eyespot. By this arrangement the eyespot casts a shadow on the enlargement when the light comes from a particular direction, and possibly affects

The function of the eyespot in Chlamydomonas 293 locomotion by a direct infiuence on the flagellum. This is not the case in Chlorophyta. In Chlamydomonas, which we may take as an example, the position of the eyespot is variable. Species have been described (Pascher, 1932) in which the eyespot is typically anterior (e.g. C. squalens), posterior (e.g. C. imitans) or intermediate (e.g. C. conjungens). It is never seen to be associated with the base of the flagella, and no connexion with the flagella has been demonstrated cytologically. Eor these reasons, although observations which may help to explain the function of the eyespot in euglenoids are of importance, caution should be used in extending this evidence to cover eyespots in other groups. Mast (1911) held that in Chlamydomonas there is no visible structure upon which the eyespot can cast a shadow, though he suggested that if it plays a part in phototaxis at all, its primary function must be the absorption of light. He later discovered, by subjecting cells to monochromatic light of different wave-lengths, that Chlamydomonas showed greatest sensitivity to a wave-length of 5030 A. (Mast 1917). This result has been confirmed by Oltmanns (1917) and Luntz (1931), both of whom found maximum response at wave-lengths near 5000 A., and this value is believed to correspond with the region of maximum absorption by the red pigment of the eyespot (Pringsheim, 1937). More advanced members of the Volvocales consisting of colonies of Chlamydomonaslike cells, also show maximum response to these wave-lengths (Mast, 1917, Gonium, Pandorina, Eudorina; Laurens & Hooker 1920, Volvox). It is interesting that many of these forms exhibit definite polarity, with the anterior part always in front when the colony is in motion. This is associated with a decrease in size of the eyespots from front to rear (Iyengar, 1933, Pandorina, Eudorina; Metzner, 1945, Volvox), which has been related to functional differentiation within the colony. It has been suggested that the cells with the largest eyespot are most sensitive to light, and largely responsible for orientation and locomotion of the colony. There is also some evidence from the behaviour of closely related organisms differing in the presence or absence of eyespots. Buder (1917) studied related races of Polytoma and Polytomella, and reported that those with eyespots were sensitive to light but those without were not. Finally, attention is drawn to a significant statement by Mast (1927) that though many species without eyespots show responses to light, no case is known of an organism with aa eyespot which does not respond. It should be clear from the brief review given above that there is considerable indirect evidence suggesting that the eyespot plays an important part in controlling phototactic responses. An opportunity to provide further experimental evidence arose during some genetical work with Chlamydomonas reinhardi. Following ultra-violet irradiation of wildtype cells, a mutant clone was isolated in which the cells lacked eyespots. A comparison of phototactic responses was made between the 'eyeless' mutant and the wild-type strain from which it originated. EXPERIMENTAL Comparison of phototaxis in wild-type and in the eyeless mutartt It was a simple matter to demonstrate that a culture of the eyeless strain in liquid medium responded to unilateral illumination by swimming either towards the light or away from it. It also appeared from experiments of this sort that the response of an eyeless culture

294 J- N. HARTSHORNE was not so rapid nor so complete as that of a wild-type culture. In order to substantiate this observation, methods were sought for obtaining more precise measurements of the phototactic responses of the two cultures. The first method was based on an experiment with Chlamydonionas described by Faminzin (1867) and amply confirmed by later workers, which showed that cells swim towards a hght source of moderate intensity but swim away from very strong light. From this observation it is reasonable to deduce that there must be some light intensity which is too high for cells to be attracted but not high enough to repel them, and an experiment was undertaken to determine this intensity for the two strains under consideration. The experiment was carried out in a dark-room, and made use of a lamp which gave a horizontal beam of white light. The intensity of the beam could be altered by means of an iris diaphragm, and the range of intensities used was further increased by varying the distance between the lamp and the culture. Light intensity at various apertures and distances was measured in foot-candles before the experiment began. The two strains to be compared were grown in liquid medium under identical conditions, and were at the same age when their responses to light were tested. Experimental procedure was as follows. With the lamp turned off, a small volume of one of the cultures was poured into a shallow glass dish which was set on a stand in line with, and at the same height above bench-level as the source of light. The glass dish was shaken gently to ensure even dispersal of the cells in the hquid, and the lamp was then switched on. When the culture had been exposed to the beam of hght for 5 min., it was examined with the naked eye. When dealing with the eyeless mutant there was usually no obvious accumulation of the cells at one side of the dish after this time, and exposure was prolonged for a further 5 min. After exposure to the beam of light, the reaction of the cells was noted, and the procedure was repeated using a different light intensity. For each repetition a fresh sample was taken from the culture, so that the conditions immediately before testing were identical in all cases. The experiment was repeated several times using cultures of different ages, and all results were similar. The wild-type strain showed distinct and rapid response to all except certain intermediate intensities, but the eyeless strain was much less responsive. Even after 10 min. exposure to the light, accumulation of the cells was not strongly marked, and there was a considerable range of intensities which caused no definite positive or negative response. A representative set of results is given in Table i. Since this first method did not give quantitative results of any precision, a second method was tried. The author is indebted to Prof. F. T. Haxo of the Scripps Institution of Oceanography, California, who devised this method and made available some unpublished data on phototaxis in gametes of Ulva (Haxo & Clendenning, unpublished). The apparatus was arranged as shown in Fig. i. A represents a lamp producing a horizontal beam of white light of variable intensity, and B another lamp giving a light intensity of 5 f.c. at the position of the absorption cell C. This cell had flat, circular sides, 22 mm. in diameter, set at a distance of i cm. apart. The cell was fixed so that it was struck by the beam of light from A with its sides perpendicular to the direction of light. In the first experiment the cell was filled with a suspension of motile, wild-type Chlamydonionas and was exposed to light from lamp B, with lamp A turned off. A dim red light was placed behind the cell so that accumulation of the organisms at either side could be seen. Red light is known to have negligible effect in causing phototaxis (Mast, 1917; Oltmanns,

The function of the eyespot in Chlamydomonas 295 1917; Luntz, 1931), and was not expected to interfere with the experiment. After 2 min. it could be seen that the organisms were gathered against the right-hand wall of the cell, that is the wall nearer the source of light. Lamp B was then switched off and at the same moment lamp A, which had been set to produce a beam of 5 f.c. intensity at the position of the cell, was switched on. This change in illumination had a striking effect on the Table i. Phototactic response to light of various intensities shown by wild-type and eyeless strains Light intensity (foot-candles) S 8 45 125 170 300 500 Wild-type Phototactic response Eyeless -1- +? (after 5 min.) + (after 10 min.) + +? (after 5 min.) + (after 10 min.) + +? (after 10 min,) -1-? (after 10 min,) Neutral? (after 5 min,) (after 10 min,) (after 5 min,) (after 10 min,) (after 5 min,) (after 10 min,) + 4- = strong positive response. + =weak positive response. = strong negative response. =weak negative response. organisms. Almost immediately they began to move towards the new source of light, and with such uniformity that a flat 'plate' of organisms could be seen to move across the cell from right to left. When the cell was observed from the side, this 'plate' was seen edge-on and appeared as a dark vertical line. As the 'plate' advanced across the cell slight differences in the speed of different individuals caused this line to become less distinct, but it remained sharply defined as far as the half-way mark. Thus it was possible to measure the speed with which the organisms travelled a distance of 5 nam. towards a light source of known intensity. In the first experiment, for an intensity of 5 f.c, the c G Fig. I. time taken by wild-type cells to travel 5 mm. was 40 sec. The procedure was repeated several times without changing the light intensity, using a fresh sample from the culture on each occasion, and the time remained constant. When the light intensity was increased, there was a slight increase in speed, and there was a corresponding decrease in speed with reduced intensity.

296 J. N. HARTSHORNE When a culture of the eyeless strain was used, it was once again found impossible to obtain accurate measurements of the phototactic responses. The procedure was as usual, and after 2 min. exposure to the light from lamp B, there was some accumulation of the organisms against the right-hand wall of the cell. The direction of light was then reversed, and the organisms began to move from right to left; but there was yery little uniformity of movement and the dark line of cell dispersed almost immediately. The same reaction occurred with all the light intensities which were tried, and in no case was it possible to measure the speed of motion. Though both experiments which have been described failed to give the quantitative comparison between wild-type and the eyeless mutant which was sought, the resuhs have a certain significance in that lack of the eyespot is not associated with complete inability to respond to hght. It has been amply demonstrated that the eyeless strain does show phototactic movements, but that the responses to light are less rapid and less uniform than those of the wild-type. This evidence appears to give some support to Strasburger's claim that the entire protoplasm is sensitive to hght, though the increased precision with which the wild-type reacts agrees well with the widely held view that the eyespot is a specialized organelle particularly concerned with the perception of light. The possibility should not be ignored that cells of the eyeless strain possess an unpigmented eyespot which has escaped notice. If this were so the feeble responses of the mutant might be due to limited absorption of hght by the eyespot, and not due to sensitivity of some other part of the protoplasm. However, the shape and size of the normal eyespot in a wild-type cell suggest that even without the pigment the basic structure would be visible in at least some of the eyeless cells. Close examination of numerous eyeless cells under high magnification has failed to show any colourless body which might represent an unpigmented eyespot. For this reason the preferred explanation of phototactic behaviour is that the eyespot is largely responsible, but that the rest of the protoplast is not entirely insensitive to light. SUMMARY A comparison has been made between the phototactic responses of normal cells of Chlaniydomonas reinhardi and those of a mutant clone in which the eyespot is absent. Lack of the eyespot is not associated with complete insensitivity to light, though the eyeless mutant reacts with much less uniformity and precision than the wild-type. It is considered that the perception of light in Chlaniydomonas is a property not restricted to the eyespot. The author wishes to thank Prof. G. M. Smith for his interest and advice during the course of this work, which was carried out in the Department of Biological Sciences, Stanford University, California. REFERENCES BuDER, J. (1917). Zur Kenntnis der phototaktischen Richtungsbewegungen. Jb. zoiss. Bot. 58, 105. ENGELMANN, T. W. (1882). Ueber Licht- und Farbenperception niederster Organismen. Pfliig. Arch. ges. Physiol. 29, 387. FAMINZIN, A. (1867). Die Wirkung des Lichtes auf Algen und einige andere nahe verwandte Organismen. jfb. wiss. Bot. 6, I. IYENGAR, M. O. P. (I933)' Contributions to our knowledge of the colonial Volvocales of South India. y. Linn. Soc. (Bot.), 49, 323.

The function of the eyespot in Chlamydomonas 297 LALTRENS, H. & HOOKER, H. D., JR. (1920). Studies in the relative physiological value of spectral lights. II- The sensibility of Volvox to wave-lengths of equal energy content. J. Exp. Zool. 30, 345. LUNTZ, A. (1931). Untersuchungen uber die Phototaxis. I. Mitteilung: die absoluten Schwellenwerte und die relative Wirksamkeit von Spektralfarben bei grunen und farblosen Einzelligen. Z. vergl. Phvsiol. 14, 68. MAST, S. O. (191 I). Light and the behaviour of Organisms. New York. MAST, S. O. (1917). The relation between spectral colour and stimulation in the lower organisms. J. Exp. Zool. 2Z, 471. MAST, S. O. (1927). Structure and function of the eye-spot in unicellular and colonial organisms. Arch. Protistenk. 60, 197. METZNER, J. (1945). A morphological and cytological study of a new form of Volvox. Bull. Torrey Bot. Cl. 72, 86, 121. OLTMANNS, F. (1917). Uber Phototaxis. Z. Bot. 9, 257. PASCHER, A. (1932). Zur Kenntnis der einzelligen Volvocalen (Beitrage zur Kenntnis der einheimischen Algenflora, I). Arch. Protistenk. 76, i. PRINGSHEIM, E. G. (1937). Ober das Stigma bei farblosen Flagellaten. Cytologia, Tokyo (Fujii Jub. Vol.), P- 234. STRASBURGER, E. (1878). Wirkung des Lichtes und der Warme auf Schwarmsporen. Jena Z. Naturw. 12, SSI- WAGER, H. (1900). On the eyespot and flagellum in Euglena viridis. J. Linn. Soc. {Zool.), 27, 463.