HOTOTAXIS AND PHOTOKINESES LITTORINA PLANAXIS Ecker May 30, 1964 Recent investigations havereported some interesting, species- specific differences in the phototactic and photokinetic responses of British Littorina (Newell, 1958; Charles, 1961). It was decided to study these reactions in one of the two Littorines common to the American west coast. L. planaxis was chosen because of its greater size and availability. The following observationsare a selection of many that were made during the period April 25 to oh The May 28, 1964 at Hopkins Marine Station in central California. Coes RESPONSES TO SUNLIGHT L. planaxis collected from horizontal rock surfaces and placed on a damp glass plate in direct sunlight invariably begin to crawl in one of two general directions. The paths of 79 snails were recorded and measured to the nearest 5 (grh 1). Since the surface of the glass is featureless, this suggests that the snails are orienting in some way to the sun itself. To test this supposi- tion the glass plate upon which a snail was actively crawling toward the sun was rotated 180. The snail also proceeded to turn 180 and again moved in the same direction as before rotation. The same experiment was then repeated with snails moving in various directions and in each case the snail turned 180 and moved in a direction approximately parallel to its original one. It has been suggested that this orientation is due to a type of "light compass reaction" in which certain directions along the 24 guve rays of the sun are prefered (Newell, 1945). However, L. planaxis from horizontal surfaces tested in the laboratory by illumination from the side proved to be entirely photonegative. Of the more than 50 snails tested, not one was observed to move toward the light for more than a short distance before turning. Thus while a limited light compass reaction may be responsible for the orientation of L. littorea, where each snail can be either photopositive or photo- negative, it does not explain the observed distribution of the ntirely photonegative species, L. planaxis. Mitsukuri (1901) reported that the Japanese snail, L. exigus was also photonegative under normal conditions and that this effect would serve to drive the snails away from the sea. To test this, the tracks of 23 snails were measured on a glass plate placed at the water's edge on beaches facing in two different directions from the sun. The observations (figures 2,3) are admittedly insuf- ficient to state conclusively whether the snails are orienting to the sun or to the sea, but an obvious predominance in the direction away from the se, as would be expected from Mitsukuri's explanation, was not observed. There remained the possibility that L. plansxis was orienting in some way to the polarized light in the sky. This type of orienta- tion has already been described for L. littorea, L. saxatalis, L. littoralis, and L. neritodies, where it was shown that these snails can orient to polsrized light by means of the intensity differences based upon Fresnel's laws of refraction. Photonegative snails were observed to orient slong the plane of polarization, and photopositive r Fiqure snsils at right angles to it (Charles, 1961). To test this, light from a 150 watt spotlight placed horizontally was reflected down- ward by a mirror and the trails of 20 snails recorded and measured to the nearest 2 under this arrangement. Examination with a polariged filter showed weak but definite polarization of the reflected light. The results are plotted on a polar graph with the 0-180 axis figure describing the plane of polsrization (gråph 4). L. planaxis, being photonegative, oriented as would be expected along the plane of polari- zation. The two snails that were crawling at right angles to this plane were then tested with artificial light from the side. They were photonegative. The control group of 20 snails was tested with nonpolarized light from above adjusted to the same intensity (500 fc). These snails did not move in any one direction long enough to enable the determination of a prefered orientation. Though a great many more snails would have to be tested in order to fully establish the nature of this orientation, one other experiment also indicated a sensitivity to polarized light. L. planaxis anesthe- tized in an isotonic MgCl, solution for one hour and then unilaterally blinded by the cauterization of the right eye invariably went into "circus movement" (Fraenkel and Gunn, 1940) toward the blinded side when illuminated by artificial light from above. Similar treatment brought no effect upon snail illuminated from above by light shining through a polaroid filter. The same lack of circus movement in polarized light has also been recorded in L. littoralis. (Charles, 1961). 27 Egten Figure VARIATION IN RATE OF CRAWLING AND TURNING IN DIFFERENT LICHT INTENSITIES Individual snails collected from horizontal rocks and dark- adapted for 24 hours were tested on a glass plate for rate of turning under varying light intensities. The light source consisted of two 150 watt spotlights from above shining through a 3" water filter. The position of the snail was marked on the glass plate itself at one-half minute intervals and the rate of change in direction determined upon later examination of the mucus trails by measuring the angle of deviation from the snail's proceeding straight path. Both right and left turns were assigned positive values. The results are summarized in Table 1. While the threshold for increased rate of turning seems to vary (see results at 350 fc), all show a greatly increased rate of turning at 1000 fc. The same apparatus was now modified by the addition of a 25 watt light at one end of the glass plate. The snails would now immediately move in the opposite direction. The speed of each snail was recorded at one minute intervals by marking the glass itself. The overhead light was switched on at 8 minutes; (see Table 2 below.) The average speed of all 10 snails is presented for each minute in graph 1. Immediately after the overhead light was turned on, there was an initial reduction in the rate of crawl between 8 and 9 minutes, and a rapid increase between 9 and 10 minutes. This reaction was consistent in 9 out of the 10 snails tested. There wes seemed te be no tendency to return to the original speed after the first few minutes of higher light intensity. Gogalor Snail 9 Rafe She/ Tatle Vesor-ly 10 P.c. 56 20 35 Table Crow 10 17 L 4 e 2.0 2.8 28 2. 2.7 2-1 2. 3 2. Regrees Tateasy 380 fe. 41 9 2° 71 32 Em per Minnle Per Re. 1000 131 2 51 570 71 mihu Te 1000 3./ 3. 2 3. 3. 3. 3 3.7 3.0 3. 7 4. 0 47 a f 20 20 t one To test whether an active incresse or decrease of light intensity is necessary for a change in rate of orawl, fifty snails were dark-adapted for 24 hours and then divided into two groups of 25 each. One group was tested in the laboratory, the other in bright sunlight. The results are summarized in Table 3. Thus it can be seen that L. planaxis behaves orthokinetically, increasing both its rate of forward crawling and rate of turning at higher light intensities. This reaction may be partly responsible for the clustering effect noted in both L. littorea (Newell, 1956 and L. planaxis (Miyemoto, 1964) unpublished). As pointed out by Fraenkel and Gunn (1940), the eventu- result of any orthokinetic and klinokinetic movement serves toking the animal to an area of lower light intensity. It is interesting to note that L. planaxis moves at less than one-half the rate reported for L. littorea. Under no conditions was observed and a snail/to move faster than 4 cm. per minute,, This rate was only approached by one snail at maximum light intensity. TENTACLE WITHDRAWAL RESPONSE Both L. planaxis and L. scutulata respond to a sudden dacrease in light intensity by a rapid but comparatively short withdrawal into their shell. Upon closer examination it was observed that this withdrawal is only partial, the foot remaining in contact with the substrate. Parker (1911) reported that locomotion in several types of gastropods is accomplished by two separate mechanisms; simple adhesion of mucus during forward motion, and actual suction when any force is exerted on the shell. To test whether this with- drawal reponse involves a change from one mechanism to another, snail was placed on a plexiglass plate with a minute hole filled with water drilled in it. As the snail erawled over the hole, a bright light from above was switched off. Air bubbles were now observed to rise in the hole, indicating an increase in suction as the tentacles and head retracted. This was observed only for a sudden but relatively small decrease in light intensity. Rapid increases in light intensity brought no similar reaction, although the sharp decrease in the rate of crawling as an overhead light is switched on (see graph 1) indicates that the snail is not entirel, unaffected by sharp increases in light intensity. SENSITIVITY TO VARTOUS WAVE LENGTHS While the tentacle withdrawal response to decreased light is consistent with newly collected snails, the response quickly- diminishes upon repeated trial. This rate of fatigue or adaptation was used to test the color sensitivity of the L. planaxis eye, or at least the color specificity of this response. Snails were illuminated from below by a 150 watt spotlight controlled by a rheostat. The snail was allowed to crawl on a glass plate placed over Corning glass filter. Light was focused on the filter by a water lens also serving as a heat filter. A second rheostat controlled 25 watt bulb also placed below the filter. This supplied enough light to observe the snail's reaction as the main light source was switched off. One red, one green, and two blue filters were tested, as well as a "control" with normal white light. In every case the intensities were equilibrated by varying the intensitjes of the 150 watt light source as measured through the filter by a thermopile and galvonometer. The light was switched off every 15 seconds and turned on 7/ seconds later. When the snail showed no reaction, in three consecutive decreasing /light trials, it was considered "adapted." No snail was used for any two trials, thus a total of 50 snails was tested. The results o are given in Table 4. A Corning Blue 4553 filter was substituted for the original sccua 4503 when analysis with a hand spectrometer showed that this first filter allowed a considerable amount of green light to pass. The slight sensitivity to red light can probably be explained by the small amount of orange or white light not absorbed by this filter. The reactions to red light consisted at best of a slight jerk of both tentacles. Thus the tentacle withdrawal response is specific only A for light in the graen portion of the spectrum. This is consistent with the findings on L. littorea, which orient only to polarized light in the blue-green portions of the spectrum (Charles, 1961). Snails retested up to 2 hours after their first adaption showed only partial and sporadic withdrawal. However, snails left on the filter at full light intensity for up to 30 minutes showed no appreciable difference in response from that of freshly collected e snails. This wouid indicate that the tentacle withdrawal response and adaptation is due to a nervous function rather than the bleaching of any specific pigment in the eye. Rafe Table o erew! Indoot5 Ouldeer) Dlnt mive 7.14 Tuhle Vanber 8lu 213 whTe 5n 12 12 10 1.5 5. 8 Wverafe W114 Goelys filler loved some Cm Rale 2.0 enfmin enfmi 2.85 Reatlens B/ue Green 3 33 21 21 35 27 3/ 20 28 33 30 25.2 a showed Perlaene 14.7 re5 hreen 171 per 19. Vumber 20 2 1 50 Rel a7 74.5 20 TAXIS AFTER EXTENDED PERTODS PHC LIGHT AND DARK ADAPTTON In the area from which the test snails were obtained, L. plana can be found both on dry rocks anditidepools. To test for any difference in phototaxis samples collected from both locations were kept in toth constant light and dark conditions for periods up to four weeks. All tests were conducted under approximately one foot of water in a standard l'x2' aquarium. A suspension of India ink was added to the sea water in the tank and a 200 watt bulb and rheostat at one end provided the light source. These conditions esteblished a good light gradient ranging from 10 fcp at the bright end to lfop in the middle. For each test 10 snails were placed facing random directions in the center of the tank. After two hour- the tank was drained and the direction of crawling established by examining the carbon-dyed mucus trail. In each case 10 snails were tested, however only the paths of the active snails are reporte here. Snails from tidepools. Of the snails tested immediately after au were collection, 7 were photopositive, 2 relatively indifferent (see Fig. 5). After 24 hours of constant light conditioning, both the light- and dark-adapted snails were photopositive without exception (see Fig. 6). Both light- and dark-adapted snails were tested every 48 hours and were, without exception, photopositive. On the 18th day, five of the dark-conditioned snails suddenly became photonegative, one re- mained photopositive. Of the light-conditioned snails, all 7 active snails were photonegative. Both the light- and dark-adapted groups had been entirely photopositive when tested on the löth day. Both groups remained photonegative until the experiment was terminated 10 days later. Snails from drx rocks. 20 k. planaris were collected from dry rocks and immediately tested. 14 of these were photonegative, 3 were relatively insensitive. After 24 hours of either light- or dark-adaption all were found to be photonegative. There is now further evidence that 24 hoursjconstant environment not only "stabilizes negative phototaxis, but actually strengthens it. Snails collected in the field and negativejgeotactic will crawl slowly upwards even when illuminated from above. After 24 hours of dark-adaption the same snail will crawl downward, although their negative geotaxis has not changed (Klabunde, 1964) unpubitsheu). Both the light- and lark-adapted snails were then tested every 48 hours and no reversal of phototaxis was observed. All remained nagatively phototacti- for the three weeks the tests were continued. In only one case were L. planaxis collected from dry rocks esifvely ever observed to be phototactic. 10 snails were kept entirely submerged in sea water under normal light conditions in the labors- tory. Tested after four weeks of submersion, 7 were photopositive and 3 moved across the beam of light. None were photonegative. Aekios This is approximately the disteibutien that would be expected from snails found in tidepools. The same apparatus was also used to test phototaxis of uni- laterally and totally blinded snails. 8 L. planaxis from a tidepoo. and 6 from thre dry rocks were dark-adapted for 24 hours and anesthetized -10- ingisotonic solution of MgCl, for one hour. The right eyes were then cauterized with a hot needle and the anesthetized snail immediately placed in a test tank. The mucus trails showed that the snails had moved and prececded in the expected direction, dry L. planaxis frem awayl and tidepool L. planaxis toward the light, with no abnormal deviation from the course. (See Fig. 7) Totally blinded snails, operated upon and tested in the same manner, proved to be totally insensitive to light and proceeded on a path closely resembling that of an unoperated snail in total darkness (Fig. 8). S. Littorina planaxis crawling on glass plates in the sunlight orient themselves primarily along the sun's rays. A response to the plane of polarization of the sunlight is the probable mechanism involved. ct L. planaxis behaves ertkokinetically, increasing both its rate of turning and its rate of forward crawling at higher light intensities. The Rittorine eye, as tested by the tentacle jerk response, is sensitive only to green light.n Kante 9 vke pad Snails found on dry rocks were always photonegative. Those found in tidepools were primarily photopositive. In either case, censTen! the response is strengthened by 24 hours of either light or darkness. Tratt ot Figure Ejure unilaTer ly Soe hormelt ad Vorae she Bided phelopes.Tive Dark Snel adeg sed Sahl:2 Figute — Frute Te1.Hy Oladet Sa ITERATURE CITED larles, G.H. 1961. The orientation of Littorina species to polarized light. Jour. Exp. Biol. 223:189. Fraenkel, F.S. and Gunn, D.L. 1940. The Orientation of Animals: Kineses, Taxes and Compass Reactions. Oxford. Fretter, V. and Graham, A. British Prosobranch Molluscs. Published for the Ray Society; Abelard and Son, Limited, Bartholomew Press, Dorking, England. 1962. itsukuri, K. 1901. Negative phototaxis and other properties of Littorina as factors in determining its habitat. Annot. Zool. Japan. 4 (1):1-19. Newell, G.E. 1958. An experimental analysis of the behavior of Littorina littorea (L.) under natural conditions and in the laboratory. J. mar. biol. Ass. U.K. 37:241-266. Newell, G.E. 1958. The behavior of Littorina littorea (L.) under natural conditions and its relation to position on the shore. :229-239. J. mar. biol. Ass. U.K. Parker, G.H. 1911. The mechanism of locomotion in gastropods. Journ. of Morph. 22: 155-170.