Four British species of Littorina, viz. L. littoralis (L.), L. saxatilis (L.), L. littorea (L.) and L. neritoides (L.), responded when crawling on horizontal surfaces in air and in sea water to plane polarized light incident from above. Photonegative winkles crawled parallel to the plane of vibration (e vector) and photopositive molluscs at right angles to the plane of vibration.
The investigations with L. neritoides confirmed the observations of Fraenkel (1927) on the reversal of phototaxis from photonegative to photopositive when this winkle was crawling in the inverted position immersed in sea water. This change of response was considerably influenced by the sea-water temperature, the optimum temperature being withip the range from 10°to 12° C.
Examination of the eye failed to reveal biréfringent structures. It is suggested that the mechanism of analysing plane polarized light is a simple reflexion/refraction phenomena based on Fresnel’s laws of refraction of polarized light, the minimum amount of light being refracted into the eye when the animal is crawling parallel to the plane of vibration.
Plane polarized light has been shown to influence the orientation of numerous invertebrates, for example the decapod crustacean Eupagurus bernhardus ((Kerz, 1950), mysids (Bainbridge & Waterman, 1957, 1958), species of Cladocera (Baylor & Smith, 1953) and numerous insects (Vowles, 1950, 1954; Carthy, 1951, 1957, 1958; Stephens, Fingerman & Brown, 1953; Frisch, 1950; Stockhammer, 1956; Wellington, Sullivan & Green, 1951; Wellington, 1953, 1955; Kalmus, 1958, 1959, to name but a few). It has also been shown that the receptors of the xiphosuran Limulus can detect changes in the plane of vibration of polarized light (Waterman, 1950, 1954a, b). More recently the ability to orientate to polarized light has been demonstrated in the marine gastropod Littorina littoralis (L.) by Burdon-Jones & Charles (1958a, b) and in Nassa obsoleta by Baylor (1959).
The investigations on Littorina littoralis have now been extended to other British species of the Littorinae, and their behaviour gives some indication of the means by which the plane of vibration of the light is detected by these molluscs.
APPARATUS AND PROCEDURE
Fig. 1 shows diagrammatically the essential details of the apparatus used. The light originated from a 500 W. spotlamp (A), passing successively through a condenser (B), a water-bath heat filter (C), a ground glass screen (D) serving as a depolarizer, and finally through a Polaroid linear polarizer (E). The latter was fitted into a rotatable housing so that the plane of vibration of the light could be altered at will. This arrangement provided an illumination of 50 ft.c. at the centre of the horizontal surface over which the animals were allowed to crawl.
The crawling surface for the trials conducted with the molluscs crawling in air consisted of a circular Perspex plate c. 40 cm. in diameter, the upper surface of which had been roughened with fine grade carborundum paste to facilitate wetting. A black non-reflecting grid ruled in centimetre squares was placed beneath the Perspex plate, the lines being clearly visible through the wetted surface.
For the trials using animals fully immersed in sea water, the Perspex plate was replaced by a circular glass trough (F) c. 32 cm. in diameter. A matt black screen (G) fitted within the trough minimized any reflexion of light from the walls. To reduce further any reflexions from the wall of the experimental cell and to prevent the regular pattern of light and dark areas produced when polarized light struck the meniscus at the margin of the water surface, a black annular diaphragm (H) was fitted to shield the walls and meniscus from direct illumination. The whole apparatus was surrounded by a tall cylindrical screen (I), blackened on its inner surface, and a wide annular mask (J) fitted horizontally around the Polaroid holder to prevent any spurious reflexions from above. All experiments were conducted in a dark room.
In a typical trial a winkle was placed facing in any direction at the centre of the crawling surface and allowed to crawl about freely until it reached the periphery. The winkles left a readily visible mucus trail on the Perspex plate surface. With the aid of the centimetre grid, facsimiles of their tracks were plotted on to a large sheet of graph paper after the completion of each trial.
The mucus trails were erased after each trial lest they should interfere with subsequent trials.
When Littorina littoralis (L.) and L. littorea (L.) were crawling immersed in sea water in the large glass trough their tracks were recorded while the winkles were in motion. Since the trials were conducted in a dark room stray reflexions from the observer were minimal and did not interfere with the illumination of the trough in any way. The results obtained did not indicate any directional bias that might have been induced by the presence of the observer.
Since the common periwinkle L. littorea (L.) may be either photonegative or photopositive (Newell, 1958a, b), the type of response was determined with ordinary light before making any trials with polarized light. This was done by removing the Polaroid from the apparatus and placing a white reflecting surface against one half of the black inner circular screen to form a cylindrical arena with one half white and the other black. With central overhead illumination photopositive winkles crawled towards the white half of the arena and photonegative ones towards the black half.
The small size of L. neritoides (L.) and the need to study its response to polarize J light while crawling upside down led to the apparatus being modified as shown in Fig. 2. The light beam was reflected upwards or downwards into the experimental chamber (F) by means of the plane mirror (M), with the Polaroid (E) interposed between the mirror and the experimental chamber. For the trials with L. neritoides in the inverted position the winkles were first allowed to attach themselves normally to the Perspex plate which was then quickly turned over and immersed in the dish of sea water.
The arrangement of screening to prevent spurious reflexions was essentially similar to that already described. The possibility of light being reflected from the observer did not arise, since mucus trails were used to record the tracks. To predetermine their sign of response to light the same technique as described for L. littorea was used. The modified apparatus was also used for the trials with L. saxatilis (L.).
ANALYSIS OF RESULTS
The reference axis employed was a predetermined horizontal direction in space related to the room in which the experiments were carried out, and assigned the bearing 0–180°. Angles were measured in a clockwise direction from o to 360°.
Experiments on Littorina littoralis (L.)
In preliminary experiments with L. littoralis (Burdon-Jones & Charles, 1958a) three experimental conditions were applied:
(a) The plane of vibration (e vector) of the light was kept parallel with the reference axis, until the animal had traversed a standard distance from the starting-point when the plane of vibration was momentarily turned through 90° and returned to its original position.
(b) The plane of vibration was rotated through 90° from the reference axis after the animal had traversed the standard distance, and left in its new position.
(c) The same as for (b) but with no Polaroid in the rotatable holder.
In these experiments the angle subtended by the end-point of the track and the reference axis was measured. The number of individuals whose tracks terminated less than 30° from the reference axis is given in column 2 of Tables 1 and 2. A large proportion of the trials fell into this category when the plane of vibration was parallel to the reference axis. Hence there is a definite tendency for L. littoralis to orientate by crawling parallel to the plane of vibration whether immersed in sea water or in air. When the Polaroid was rotated through 90° a consequent turn of 90° was induced in the mollusc, as can be seen from the higher figures in column 4 than in column 2 of Tables 1 and 2, for the second part of group B. Application of the χ2 test showed the results to be highly different from random expectation. In applying this test, it was assumed that one third of those that reached the periphery would be expected in each of columns 2–4.
The response of L. littoralis to polarized light of different wavelengths was investigated by using a series of Chance colour filters (K) placed between the depolarizer and the Polaroid. For these trials and subsequent trials with polarized light on the other species, the tracks were analysed in a slightly different manner and in more detail.
The resultant angle made by each centimetre of track was measured. Those parts of the track which were approximately parallel to the reference axis were then estimated by counting the number of centimetres where the track lay within the sectors 0–30°, 150–180°, 180–210° and 330–360° on either side of the reference axis. They were expressed as a percentage of the total distance crawled. They were then compared with the percentage of the track which lay in the sectors 60–90°, 90–120°, 240–270° and 270–300° on either side of the axis at right angles to the reference axis. Table 3 is given as an example of this method of tabulating the results. It shows the responses of L. littoralis to polarized light within the range of wavelength 3500–6500 Å. The results were analysed statistically by applying Student’s t test to ascertain whether the mean difference between the pairs of the percentages of the two equal angular groups was significantly greater than zero (Davies, 1949, t test difference method). [In conducting these experiments with polarized light of different wavelengths on L. littoralis, which had already been found to orientate parallel to the e vector (Burdon-Jones & Charles, 1958a, b), we were only interested in whether the tracks parallel to the plane of vibration exceeded those at right angles to it. As a difference in a particular sense was expected a single-tailed probability curve was employed for Student’s t test. This was done by halving the probability value P obtained from the ordinary tables for t.]
This method was employed for all subsequent trials on Littorina species. Table 4 summarizes the results of the trials of L. littoralis to polarized light of different wavelengths. When light was transmitted through the blue-green filter (3500 and 6500 Å., first two sections of Table 4), L. littoralis showed a significant tendency to align itself parallel with the plane of vibration of the light. The t test gave P < 0·01 in both sets of experiments. However, trials with the other three colour filters decreased the efficiency of orientation, between the distance traversed parallel to the plane of vibration (column 2) and the distance crawled at right angles to this plane (column 3).
No attempt was made to equate the light energies for each wavelength in these comparative trials, but the relative intensities were of similar order. The relative intensities of the light to that of the standard apparatus without colour filter (50 ft.c. at the centre of the crawling surface) was obtained from the transmission curves for the filters used and the spectrum of the tungsten filament light source. These were as follows: blue-green, 11 ft.c.; green, 11 ft.c.; orange, 21 ft.c.; and red, 10·5 ft.c. at the centre of the crawling surface.
Thus the results showed that L. littoralis was capable of orientating to plane polarized light between wavelengths of 3500 and 6500 Å. (blue-green filter) and of relatively low intensity (11 ft.c.), but orientation was less pronounced at other wavelengths despite the fact that these were of similar orders of intensity and, in the case of the orange filter, considerably higher (21 ft.c.).
Experiments on Littorina saxatilis (L.)
The interesting observations of Fraenkel (1927) on the reversal of phototaxis from photonegative to photopositive in L. neritoides while crawling inverted under sea water prompted a similar investigation (using the apparatus shown in Fig. 2) with L. saxatilis, since in some localities the habitats of the two species are almost identical. No analogous phenomena were apparent. L. saxatilis gave the same response as L. littoralis and orientated parallel to the plane of vibration whether upright or inverted. Both species were invariably photonegative when tested in the black and white arena. The results of the trials with L. saxatilis are summarized in section A of Table 5.
The results of trials of both the upright and inverted individuals showed that a greater proportion of the distance crawled by L. saxatilis tended to be parallel with the plane of vibration (column 2) than at right angles to it (column 3). The results were significant at the 0·05 level for the inverted trials. No doubt a higher degree of significance would have been obtained for both sets of trials if the number of trials had been larger.
Experiments on Littorina littorea (L.)
In contrast to L. littoralis and L. saxatilis, L. littorea is irregular in its response to light (Newell, 1958b), being sometimes photopositive and sometimes photonegative, but rarely or never indifferent. At the time these experiments were conducted on L. littorea, the air temperatures were in the region of 18–20° C. and the animals behaved very sluggishly except when immersed in sea water. Therefore a series of trials was carried out with the animals under water as well as in air. The results are summarized in section B of Table 5. They show that both in air and under water photonegative L. littorea behave in a similar manner to L. littoralis, the percentage distance crawled within a 30° sector from the plane of vibration (column 2) being higher than that within a 30°sector of the plane at right angles (column 3). The t test gave values of P < 0·3, > 0· 2 for the trials in air and P < 0·05, > 0·02 for the trials under water. Photopositive L. littorea, both in air and under sea water, orientated just as efficiently but at right angles to the plane of vibration, the distance travelled within a 30° sector of this plane (column 3 of section B) being much greater than the distance traversed within a 30° range either side of the plane of vibration (column 2). The values of P determined by the t test for these photopositive periwinkles were highly significant for both sets of trials (P < 0·01, > 0·001 ; and P < 0·001).
Newell (1958b) noted that on horizontal surfaces L. littorea tended to loop back and forth on its tracks when orientating to the sun’s position. Similar looping movements were noted in many cases when this species orientated itself in relation to the plane of vibration of polarized light.
Experiments on Littorina neritoides (L.)
The reversal of the light responses of L. neritoides reported by Fraenkel (1927) suggested that its reaction to polarized light should be tested when crawling normally and when crawling upside down. At water temperatures in the region of 20–22° C. their responses to light were erratic, but in sea water cooled to any temperature between 10 and 12° C. their behaviour was more consistent.
The results are summarized in section C of Table 4. Like L. littorea, photonegative L. neritoides crawled parallel to the plane of vibration when they were upright or when inverted, the greater proportion of the distance travelled being within a 30° sector of the plane of vibration (column 2). Like photopositive L. littorea, photopositive L. neritoides crawled at right angles to the plane of vibration, the greater percentage of the distance traversed being within a 30° sector each side of a plane at right angles to the e vector (column 3) than in the corresponding sectors parallel to this plane. The results of trials with the animal upside down were fairly conclusive (P values < 0·2, > 0·1 ; and P < 0·05) as were the trials with photonegative winkles crawling in the upright position (P < 0·01, > 0·01). The negative results obtained with photopositive winkles when crawling upright were probably the outcome of insufficient trials.
POSITION AND STRUCTURE OF THE EYES
The eyes in the family Littorinae are relatively small structures (about 240μ along the long axis of the optic cup in an adult L. littoralis) embedded in the outer sides of the tentacles at about one-third the distance from their bases. In Fig. 3 the angular settings of the eye in relation to the tentacle and longitudinal axis of the mollüsç are shown. The optic cup is set obliquely with the lens aperture facing upwards, outwards and slightly anterior. Directly above and extending over the outer side of the eye the external layers of the tentacle are transparent and afford the eye a wide field of vision dorsolaterally.
It is apparent from Fig. 3 that with the optical axis of the eye subtending an angle of c. 40°with the vertical axis of the winkle (Fig. 3C) and c. 45°with the longitudinal axis (Fig. 3A) considerable depression of the head and tentacles is required before the eyes can receive any light reflected from the substratum close at hand or from the foot of the winkle, despite the slight forward tilt of the optic cup from the vertical axis (Fig. 3B).
In shape the optic cup is ellipsoid with a slight flattening frontally in the region of the lens aperture. The region between the lens and the retinal cup is filled with a clear gelatinous substance. A layer of dense black pigment surrounds the retina and encloses the whole of the optic cup with the exception of the lens aperture, effectively shielding the retina from any light except that admitted by this aperture.
The lens was examined under a polarizing microscope and by sectioning. It was seen to consist of a series of concentric spherical laminae which showed no rotation of the plane of vibration of polarized light when viewed through crossed nicols. The eyes of the other winkles studied were similar in structure.
From the results for the four species of winkle investigated, it is reasonable to correlate the response to plane polarized light with the simple response to light and shade. Photonegative winkles invariably orientated parallel to the plane of vibration, and photopositive winkles at right angles to it. These facts, together with the absence of birefringence in the lens structure, point to a simple non-birefringent mechanism for the detection of the plane of vibration of the light. Such a mechanism was put forward by Stephens et al. (1953) for the orientation of Drosophila to polarized light, and was also favoured by Baylor & Smith (1953) for the orientation mechanism of Daphnia and other Cladocera. It was also listed among the possible analysing mechanisms by Waterman (1953, 1955), Stockhammer (1956), Bainbridge & Waterman (1957), Kalmus (1959) and favoured by Newell (1958b) as the probable means of orientation for L. littorea.
This mechanism is based on Fresnel’s laws of refraction of polarized light. These are concerned with the percentage of light refracted at the interphase of two none birefringent media, expressed as a function of the angle of incidence, the indices of refraction of the media and the plane of vibration.
With reference to Fig. 4A and B, let Io represent the intensity of a beam of plane polarized light that is incident at an angle i at the interphase between two media whose indices of refraction are n and n1 respectively. Let r represent the angle of refraction and I the intensity of the reflected beam. The proportion of light reflected, provided there is no loss by scatter, will be I/I0 and the proportion refracted will be 1 – (I/I0).
When the plane of vibration of the incident light is perpendicular to the plane defined by the incident and reflected rays (Fig. 4A), Fresnel showed that
The angular settings of the flattened region of the lens aperture and optic cup in the Littorinae are in the ideal positions for these phenomena to occur (Fig. 3).
When the head of the mollusc is parallel to the plane of vibration (e vector) of polarized light coming from above the animal, as shown in Fig. 5 A, the plane of vibration is perpendicular to the plane of the rays incident and reflected from the eye surface. By Fresnel’s laws the minimum amount of light is then refracted into the eyes.
Conversely when the head is at right angles to the plane of vibration (Fig. 5B), the maximum amount of light is refracted into the eyes. Consequently, as the winkle turns its head to align itself parallel with the plane of vibration, less light is refracted on to the retina, simulating the effect of moving towards or into the shade.
As a result of the exploratory movements of the head a photonegative animal will orientate its body and move in a direction which results in the minimum amount of light being refracted into its eyes, i.e. along the plane of vibration. Conversely if photopositive, the animal will move at right angles to the plane of vibration so that the maximum amount of light will enter its eyes. Although exploratory movements of the tentacle are mainly executed by the distal portion, some movement of the basal portion bearing the eye does occur. This, together with swinging movements of the head, will enable the eyes to move through a considerable angle and so detect changes in the amount of light received by the retina.
The concentric laminae of the lens of the eye in these animals would tend to accentuate any reflexion/refraction effect. Increasing the number of refracting layers will increase the number of times that the relative differences between the intensities of the reflected and refracted light are altered, since at each phase boundary the Fresnel effect will operate in proportion to the values of the refractive indices of n1/n2, the two phases. This would increase the effect of light and shade experienced by the winkle when it moved the plane of the eye surface in relation to the e vector of the polarized beam.
I wish to thank Dr C. Burdon-Jones for his help and advice at all times in the supervision of this research, and Dr D. J. Crisp for many helpful suggestions, for his consideration of the statistical problems involved and for the concluding formula derived from Fresnel’s laws.