Orientational Movements of the Foot of Littorina Species in Relation to the Plane of Vibration of Polarized Light

There is no doubt that extraocular analysis plays a part in the orientation of many animals to plane polarized light. Such an analysing mechanism is usually based on the light intensity gradients produced when polarized light is reflected from a suitable substratum and its importance has been stressed by several workers (Baylor & Smith, 1957, 1958; Baylor & Kennedy, 1958; Bainbridge & Waterman, 1958; and more recently by Baylor, 1959, and Kalmus, 1958, 1959).

Baylor (1959) showed that the orientation of Nassa obsoleta depends entirely on extraocular analysis, but Charles (1961a) believed that Littorina littoralis (L.) (not L. littorea (L.) as misquoted by Baylor, 1959) was capable of detecting the plane of vibration of polarized light, both by means of substrate reflexions and direct perception through the eyes, because shading the eyes from incident polarized light diminished the response.

It was felt desirable to demonstrate a direct response to incident polarized light by removing surface reflexions altogether. An entirely new technique and form of apparatus was therefore devised.

The essential features of the new apparatus, shown in Fig. 1, were the use of fixed animals and the observation of orientational movements of the winkle’s foot to determine its intended direction of crawling.

The winkle was held rigidly by a supporting rod (F) so that its foot rested on a small ball of paraffin wax (G) of such a size (c. 1 in. diameter for Littorina littoralis) that it could be rotated uniformly with the minimum of friction. The ball was floated on a column of sea water in the glass-bottomed cylinder (H), and the height of the water was adjusted so that no upward pressure was exerted on the animal’s foot. Both the ball and surrounding cylinder were sufficiently small not to be included in the winkle’s downward field of view. The surface of the ball was painted a matt white and uniformly stippled with small black dots. Any crawling movements executed by the foot of the animal were directly translated into a rotary movement of the ball. The dots made the direction of rotation of the ball readily apparent.

Forward crawling movements of the foot produced a rotation of the ball such that its upper surface moved in an antero-posterior direction in relation to the long axis of the animal. Likewise, when the winkle attempted to turn to the left or the right, the unequal pattern of muscle contractions in the foot, which normally brought about the change of direction, resulted in a clockwise or contra-clockwise rotation of the ball.

Enough light passed between the ball and the rim of the cylinder (H) to illuminate the bottom hemisphere of the ball, an enlarged image of which was viewed against the eyepiece graticule (L) via a system of mirrors and lenses (I), (K) and (M). A ‘Pointo-lite’ was used as the source of illumination to reduce stray reflexions from the walls and floor of the dark room in which all trials were conducted. The light was adjusted so that the winkle was illuminated by a parallel beam of plane polarized light c. 3 cm. in diameter and 58 ft.c. in intensity, the minimum amount being allowed to pass beyond the cylinder (H). The amount of this stray light reflected from the blackened floor (O), tube (J) and cylindrical screen (N) was found by experiment to be negligible in comparison with the amount of light directly incident upon the winkle (see below). As an extra precaution the rim and exterior of the cylinder (H) were painted matt white. Polarized light reflected from a matt white surface is largely depolarized, and most of the reflected light is scattered. In contrast a matt black surface, although reducing the amount of light reflected, would give this light a far more pronounced directional bias in relation to the plane of vibration of the incident rays (Kalmus, 1958).

The relation between the winkle’s size and the diameter of the wax ball was fairly critical. Smaller specimens required a correspondingly smaller ball and supporting cylinder.

A line parallel to the fixed longitudinal axis of the winkle’s shell and designated 0-180⁰ was taken as the reference axis. The various settings of the plane of vibration of the light employed in the trials were referred to this axis (Fig. 2). The 0−180° setting was chosen to give the mollusc a symmetrical stimulation, and according to the Fresnel effect a photonegative winkle should endeavour to crawl parallel with the reference axis. Conversely, the −90°, + 90°position of the plane of vibration should evoke a similar response in photopositive individuals. The 45°, 135° and 60°, 120° settings were selected to produce an asymmetrical stimulation and induce turning responses.

In a typical trial a winkle was attached to the adjustable supporting rod with the longitudinal axis of its shell parallel to the reference axis. The phototactic sign of each winkle was determined before and after each set of trials by illuminating it with a horizontal beam of ordinary light, first from one side and then the other at 90°to the reference axis. This produced a turning response away from or towards the light source and a corresponding rotation of the ball. The sequence of trials with various treatments and controls were conducted in a random manner to overcome any possible effects induced by fatigue and habituation.

The results were recorded in the following manner. The winkle was illuminated with polarized light and the Polaroid set at one of the positions illustrated in Fig. 2. Each trial consisted of a fixed number of time periods, and the direction of rotation of the ball was recorded at the end of each period, which was indicated by an audible time signal. 40 periods of 10 sec. duration constituted one trial (except in the initial trials with L. littoralis when each trial comprised 20 periods of 20 sec. duration) and the results classified under three heads :

1. ‘Neutral’ rotation of the ball, mollusc not attempting to turn. (N, Tables 1−4.)

   Table 1.

   Responses of Littorina species to plane polarized light with substrate reflexions reduced to a minimum

   

   

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2. Clockwise rotation, indicating left-hand turning. (L, Tables 1−4.)

3. Anticlockwise rotation, indicating right-hand turning of the winkle. (R, Tables 1−4.)

This enabled the relative proportions of the turning responses to be expressed numerically.

The neutral rotations were ignored and the number of left-and right-hand turns from each set of trials for one particular Polaroid setting were totalled, arranged as a contingency table and tested for significance by the χ² test. The expected value (null hypothesis) was in all cases assumed to be 50 % in each direction of rotation, and allowance was made for continuity (continuity correction for the χ² test employing one degree of freedom, Davies, 1949).

It was necessary to apply two different forms of the χ² test according to the form of experiment.

When the plane of vibration was set parallel to or at right angles to the reference axis (Fig. 2) any movement of the head and tentacles to the left or to the right of the reference axis would produce the same small change in intensity of the light refracted into the eyes, since the effect of turning the head in either direction was symmetrical. Consequently, turning movements would be expected to occur with equal frequencies in either direction. Therefore the two tails of the probability curve should be used, the probability of the null hypothesis, viz. that there was no bias in either direction of turning, being given by the normal χ² tables, for one degree of freedom.

When the plane of vibration was set at any other angle, e.g. − 45°, +135°, to the reference axis (Fig. 2), a movement of the head and tentacles to the left would be expected to diminish the amount of light refracted into the eye, whereas a movement to the right (e.g. − 40°, +140°) would be expected to increase it. Hence the anticipated effect would no longer be symmetrical. It is therefore possible to predict the direction of turning left or right, which should predominate in a photonegative animal for − 45°, +135° or any of the correct directions. In this case, therefore, we are interested only in whether the frequency of turning in the predicted direction can be accounted for by chance. If the frequency of turning predominated in the opposite sense to that expected the theory would be wrong. We therefore should use the singletailed test with half the probability value given by the standard tables for χ².

Table 1 summarizes the results obtained for L. littoralis (L.), L. saxatilis (L.) and L. neritoides (L.). All the specimens used were photonegative.

With the plane of vibration at 0-180° (treatment A) the frequency of ‘neutral responses was in excess of the frequency of the sum of the responses to the left (columns L) and to the right (columns R). These latter were of approximately equal frequency and the χ² test showed no significant difference. This result is interpreted as an attempt to crawl along a line parallel with the plane of vibration.

When the plane of vibration was at − 45°, +135° (treatment B) the proportion of neutral rotation fell and the amount of turning to the left (columns L) was significantly greater than to the right (columns R). The χ² test gave a highly significant value of P < 0 · 0001 for all species. The winkles were therefore trying to align themselves parallel with the plane of vibration of the light (cf. Fig. 2).

The results for treatment C with the plane of vibration set at + 45 °, −135 ° were exactly similar with a significant turning response to the right.

Similar results were also obtained with the Polaroid settings − 60 °, +120 ° (treatment D) and + 60 °, − 120 ° (treatment E).

When the plane of vibration was set at − 90 °, 4+90 °, i.e. with the plane of vibration at right angles to the winkle’s longitudinal axis and with symmetrical stimulation, there was no significant difference between the left-and right-hand turning responses (columns L and R). The winkles performed alternate twisting movements throughout 90° in an effort to align themselves parallel to the plane of vibration, and it can be seen that the frequency of neutral responses was much lower than in treatment A.

As previously described (Newell, 1958; Charles, 1961b), L. littorea exists in either a photopositive or photonegative phase. Table 2 summarizes the results obtained using fixed animals of this species which had previously been tested for their sign of response to light.

Photonegative specimens behaved in an identical fashion to L. littoralis by trying to orientate parallel to the plane of vibration (treatments A-D, Table 2).

The photopositive L. littorea (treatments A-D, Table 2) showed the same results as those previously obtained for freely moving photopositive periwinkles (Charles, 1961 b) in trying to orientate at right angles to the plane of vibration.

The results for each of the treatments in Table 2 using photopositive L. littorea were the converse of those obtained with the photonegative periwinkles, using the same settings of the plane of vibration.

The eye shields used to shade the eyes of the winkles from the incident polarized light were identical with those previously described and used with freely crawling L. littoralis (Charles, 1961a). These shields, although shading the winkle’s eyes, still permitted enough light to fall on those parts of the apparatus directly beneath the winkle. This enabled an experimental assessment of the efficiency of the apparatus in restricting stray reflexions to regions outside the winkle’s downward field of view.

Table 3 summarizes the results obtained. The control treatments in which the eyes of the winkles were unshaded produced results identical with those obtained in the previous trials with L. littoralis and already given in Table 1.

The photonegative specimens invariably attempted to align themselves parallel with the plane of vibration of the light when the latter was set at various angles to the reference axis (treatments B-E). In each case the asymmetrical stimulation produced a significantly greater tendency to turn to one side (right or left) more than the other. The values of P were all < 0·001, and when summed together, allowing for the signs of rotation of the Polaroid, the results were overwhelmingly significant (Table 3, Summary Table).

When the same specimens were tested with their eyes shaded from all incident polarized light from above, there was a general increase in the amount of ‘neutral’ movement for each of the treatments B-E. Furthermore, there was a complete absence of response to the plane of vibration. Even when all the results were combined, allowing for the signs of rotation of the Polaroid, the results were definitely not significant (Table 3, Summary Table).

Thus with this form of apparatus, shading the eyes completely destroyed the ability to orientate to the plane of vibration. Since it had been previously demonstrated that L. littoralis with shaded eyes could orientate to polarized light by means of the intensity gradients in substrate reflexions (Charles, 1961 a), it is contended that this apparatus effectively reduced such reflexions to a minimum.

Therefore L. littoralis is capable of orientating to polarized light directly incident upon the eyes.

Summary Table. Summary of all the results with treatments B, C, D and E

Fresnel’s laws of refraction of polarized light state that at all angles of incidence, save o° and 90°, light vibrating in the plane defined by the incident and reflected rays will be more efficiently refracted than light vibrating in the plane at right angles to this. The difference in intensity of the refracted light caused by the angle subtended by the plane of vibration decreases progressively and finally disappears when the angle of incidence of the polarized rays becomes zero (Stephens, Fingerman & Brown, 1953; Waterman, 1954).

The theory previously put forward to account for the orientation of the Littorinae to polarized light, based on Fresnel’s laws of refraction (Burdon-Jones & Charles, 1958 ; Charles, 1961b), predicts therefore that if the angle of incidence of the polarized light on the surface of the eyes were 0° no orientation should be possible.

The apparatus and technique employing fixed animals and a rotating wax ball is particularly suitable for testing this prediction.

The apparatus illustrated in Fig. 1 was modified by the insertion of two pivots in the supporting framework of the lamp, mirror and Polaroid housing. These pivots were set in such a way that the light beam could be inclined at an angle to the sagittal plane of the winkle, by being moved through an arc the plane of which was at right angles to the saggital plane.

For the experimental treatments the beam was tilted to subtend an angle of 45°to the vertical axis of the mollusc. The angle of incidence of the greater part of the eye surface of the mollusc would then be of the order of only 5°. The beam would still be centred on the mollusc whether directed vertically or inclined.

Since tilting the beam would inevitably cause unequal illumination of the eyes, unilaterally blinded L. littoralis were employed in these trials. The entire left or right eye was excised from winkles which had been previously narcotized with a 7·5 % solution of magnesium chloride in sea water. After a period of 2−3 days the wounds healed and all molluscs made a complete recovery. These animals were not entirely normal in behaviour and tended to circle towards the blind side; they could, however, still discriminate between light and shade and could locate small dark areas in an illuminated arena.

Winkles with their left eyes excised were tested with the beam vertical and tilted so that their right eyes were illuminated ; and those blinded on their right-hand sides, mutatis mutandis.

Otherwise the procedure for the trials and the method of recording and analysing the results were the same as described in the previous section.

The results are summarized in Table 4. The results from the trials with winkles blinded on their left and on their right side were exactly symmetrical. Therefore only those results for animals blinded on the left side will be considered in detail.

When the plane of vibration was set at 0−180° (cf. Fig. 2) and the light beam vertical (treatment A, α, Table 4) the winkles behaved as normal animals and endeavoured to crawl parallel to the plane of vibration. There was no significant difference between the frequency of left-and right-hand turning and there were no circus movements. Freely moving unilaterally blinded animals were also free of circus movements in the plane polarized light.

With the beam tilted, however, but with the plane of vibration still parallel to the long axis of the mollusc, the frequency of turning to the right tended to be significantly greater than that to the left (treatment A, β, Table 4). This result was surprising, and the reason for it is not at present understood.

With the plane of vibration set at − 45°, +135° and the light beam vertical (treatment B, α) a significant turning response to the left was obtained in the expected direction (P < 0·001). However, on decreasing the angle of incidence by tilting the beam (treatment B, β) the efficiency of orientation decreased and there was little difference between the two possible turning responses (columns L and R) (P < 0·5, > 0·1).

Conversely, when the Polaroid was set at +45°, −135° (treatment C, α) a significant turning response was again obtained in the expected direction with the beam in the vertical position, but the effect disappeared when the beam was tilted to 45° and the angle of incidence decreased (treatment C, β).

These trials show conclusively that L. littoralis, L. saxatilis, L. neritoides and L. littorea can, in the absence of the pattern of light intensities produced when plane polarized light is reflected from a suitable substrate, orientate directly to the plane of vibration of the polarized light incident upon the winkle from above. Photonegative winkles will orientate themselves parallel with, and photopositive winkles at right angles to, the plane of vibration (e vector) of the light.

When the angle of incidence of polarized light falling on to the surface of the eyes of L. littoralis is reduced towards zero, the ability of the winkle to orientate to the plane of vibration disappears. It is a reasonable supposition that this is brought about by diminishing the differences between the intensities of light refracted into the optic cup relative to the plane of vibration, and these results are put forward as further proof of the reflexion/refraction mechanism of orientation. These results afford an interesting comparison with those obtained by Waterman (1954) with the angle of stimulus incidence of plane polarized light on single ommatidia from the compound eye of Limulus. He found that when the rays of polarized light entered an ommatidium parallel to its optical axis and normal to the corneal surface, little or no polarized light sensitivity was obtained. This was in contrast to the ability of the ommatidia to detect the plane of vibration when the angle of incidence of the rays was large and struck the surface of the cornea at an oblique angle. Waterman (1954) suggests the possibility of a Fresnel reflexion/refraction mechanism in the polarized-light sensitivity of Limulus.

An interesting feature of the experiments with L. littoralis was that unilaterally blinded winkles were able to give an equally good response to the plane of vibration of a vertically directed beam as winkles with both eyes intact. This fact, together with the observation that unilaterally blinded winkles can orientate to a small dark area, shows that orientation does not depend on balanced stimulation of the two eyes. It seems likely that the recognition of the plane of vibration of polarized light and of light and dark areas of the visual field is achieved by the changes in light intensity produced on the retina as the animal swings the head and tentacles from side to side.

Since the Fresnel reflexion/refraction phenomena occur at the interphase between the eye surface and the external medium, it is debatable whether the term intraocular perception can be used in this context. However, the concentric laminae of the lens provide numerous interphases which contribute to the Fresnel effect (Charles, 1961 b) and in this sense the mechanism is partially intraocular. Some distinction should be made between this method of analysing polarized light and purely extraocular perception, such as responses to brightness patterns in substrate reflexions.

The role, if any, that polarized light orientation plays in the life of winkles on the shore has yet to be investigated. It is theoretically possible for the winkle to respond to polarized light from the sky, but responses to reflected substrate patterns may be of greater importance. Reflected patterns of polarized light have their maximum brightness in the same azimuth as the sun and these could play a part in the light compass/ sun orientation responses exhibited by the Littorinae.

My thanks are due to Dr C. Burdon-Jones for his help and advice at all times in the supervision of this research. I am also grateful to Dr D. J. Crisp for many helpful suggestions including the initial ideas leading to the rotating ball technique and his consideration of the statistics. I would also like to thank Dr O. L. Davies of the I.C.I. Pharmaceutical Division, Alderley Park, Macclesfield, Cheshire, for his advice with some of the statistical problems.