Responses to Light in the Gastropod. Olivella biplicata; evidence of a Circadian Ehythm by Margaret A. Stetson Hopkins Marine Station of Stanford University Pacific Grove, California 93950 DUCTION INIHO The burrowing snail, Olivella biplicata (Sowerby, 1825), inhabits the intertidal and adjacent subtidal zones on sandy shores along the wastern coast of North America. Edwards (1969) noted that the animals tend to burrow under the sand surface when exposed to light, and, in experiments with light gradients, found that larger snails tend to cluster at lower light levels than smaller snails. David Phillips of Hopkins Marine Station had also observed that Olivella tend to be out on the sand surface at night and burrowed below it during the day, and suggested that a biological clock might be involved (pers. comm., 1972). The present study has been undertaken to further investigate this response to light and to examine the possibility of circadian rhythms in Olivella biplicata. All studies were carried out in the period April - June, 1972, on populations available at low tide at the south end of Monterey Bay, California. Margaret Stetson pr FTELD STUDIES An initial field study was carried out to see whether more snails were on the sand surface at night than during the day. Observations were made on consecutive days at low tide in three areas: 1) the exposed portion of the beach (Boatworks Beach) between Mussel Point and Point Alones, Pacific Grove, 2) a sheltered sandy cove on this same beach, and 3) the beach just east of Fishermans Wharf (Wharf #1), Monterey. The populations observed were always those covered by approximately one foot of water at the time of observation. At each sampling period, a 0.25me sheet metal quadrat frame was pushed into the sand in two different places in each area, and the numbers of animals above and below the sand within the frame were recorded. Illumination during night studies was provided by a five-battery flashlight shielded by two layers of red cellophane. The results are shown in Figure 1. The snails found ranged from 1.5 to 3.Ocm in shell length. At least 50% of the observed populations were below the surface at any one time. Almost the only individuals found above the sand surface in direct light were mating pairs, suggesting that the stimulus of mating may supercede the stimulus to light. Excluding mating pairs, data from three daytime readings (April 22, 23, and 24) and three nightime readings (April 24, 26, and 27) were pooled and averaged; the dillerence between mean percentages of animals above the surface at night and during the day was significant at the 998 level. The small samples found at Margaret Stetson night on April 23 and 25 are not included in this analysis; they reflect the relatively high level on the beach at which it was necessary to work due to tide and sur Additional observations were made on a population of 1000 Olivella biplicata, 1.5 to 3.Ocm in shell length, taken on May 2 from Monterey Harbor and maintained on a simulated beach composed of two tons of sand in an outdoor tank, 15.5 x 9.5 x 2ft. at Hopkins Marine Station. This beach had a slope of approximately 8' and the tank received a constant flow of fresh sea water. Low tide was simulated by slowly draining and refilling the tank every day between 0800 and 1400hrs; at low tide (1100 hrs.) the tire beach was uncovered. The daylight low tide was unfortunate, since it introduced another environmental variable during daylight, but was unavoidable since the tank was also in use by other investigators. Observations began one week after introduction of Olivella to the tank, and were continued at varying intervals over a six day period. The results of this study are displayed in Fig. 2. The difference between the number of animals at the surface during daylight (0600 - 2000 inclusive) and those up at night, pooling data for the whole period, is statistically significant at the 99% level. However, this includes the combined effects of light and low tide during the day vs high tide and darkness at night. By excluding all daylight observations taken after 0800 and before 1400, day vs night high tide conditions can be compared. "Using these data, the difference between the average mber of animals up during the day vs those up at night is still significant at the 99% level. There seemed to be differences Margaret Stetson in activity between sunny periods and periods of fog or dusk on May 9(0800 and 1600), May 10( 1800 and 1600), and May 16 (0800 and 1200). These suggest the orthokenetic light response postulated by Edwards (1969). Were this experiment to be repeated, more useful data could be obtained by a) elimination of tidal vatiations, and b) setting observation times at critical periods, eg. noon, midnight, and one hour before and after sunrise and sunset. r LABORATORY STUDIES In order to quantify the effects of light on activity of Olivella biplicata and to determine the existence of a biological clock, several laboratory experiments were undertaken. In all experiments, animals were kept in 17.5 x 10.5 x 10 in. aquaria, supplied with fresh running seawater at about 1'C above ambient sea temperature. Simulated daylight was provided by four General Electric flourescent lights (F48712 Cool White, High Output), suspended 16 inches above the aquaria. Animals were xamined periodically; readings in darkness were made in red T7 light using a Starlite Illuminator with link and variable transformer (A0 - model 865, 115 VAC, 60 cycles, 2.75 amps) set at 5.5v, using a G.E. (1480) bulb screened by a Corning filter, number 243(minimum transmission at 610 nm, midpoint at 630 nm, peak at 660 nm). This light source was shown in preliminary tests to have no effect on the activity of the subjects. All animals used were 1.5 to 3.Ocm in shell length. Margaret Stetson Locomotor activity in light and darkne The larger numbers of animals above the sand surface at night suggests greater, or at least more wide-ranging, locomotor activity during darkness. To see whether this was the case, a comparative study of displacement under conditions of light and dark was made. Fifty individuals were collected at lower low water on May 8 at the sheltered cove of the Boatworks Beach. These were divided between two aquaria, one with constant flourescent illumination, the other kept in darkness. Each tank was covered on the bottom with sand to a depth of three inches, and was partitioned into a labyrinth of four compartments or boxes, incompletely separated by vertical sheets of glass (Fig. 3, top). All animals were initially placed in Box 1 of each tank, and their subsequent positions were checked at intervals over the next three hours. In the lighted tanks, animals could be detected by the positions of their siphons above the sand, In the dark tanks it was necessary to locate the burrowed animals by "feel". The results indicate clearly that displacement with time was greater in the dark tanks than in those in the light. The number of animals remaining in Box 1 after 3.5 hrs was significantly greater (958 level) in light than in the dark. The experiment suggests that, in darkness, not only are more snails found on the sand surface, but their locomotor activity is greater. There is some possibility that "feeling" in the dark tanks stimulated these animals to greater movement, and that the greater displacement here might be due in part to this. Should Margaret Stetson this experiment be repeated, I suggest using a shallower layer of sand on the bottom, and perhaps providing each snail with a trailing marker which would project above the sand, Experiments with circadian rhythms: The presence of a fairly consistent rhythm of activity with respect to light and dark suggests the possibility of a biological clock. Several experiments were set up to test for the presence of a circadian rhythm and to attempt entrainment by an artificial light-dark cycle. Constant darkness Two sea water tanks, both provided with approximately three inches of sand from the area of sample collection and with continuous flows of fresh sea water, were set up in a darkroom with alternating periods of light and darkness. The light period for both tanks corresponded approximately to that of a normal day, with lights on at 0800 and off at 2000 hrs. Each tank received 70 Olivella, collected at lower low water on April 30 from the protected sandy cove on the Boatworks Beach. After 6 days, one tank continued as a control, with alternating 12 hr periods of light and dark, while the other was left in constant darkness. Observations were continued for another 6 days. During the entire 12 day period tanks were observed at intervals, both day and night, and at each observation the number of animals at the surface (both mating and single animals) was recorded. The results are shown in Fig. 4. For the first 6 days, both tanks show clear daily cycles of activity correlated with Margaret Stetson the light regime. For both tanks, the difference between the average number of animals at the surface by "day" and at night. differs significantly (98% level, using the Yates correction for X and excluding mating pairs). The dark period, in this and subsequent analyses, was considered to include observations from 2100 to 0800 hrs and the light period from 0900 to 2000 hrs. since readings at 0800 and 2000 hrs were made just as the lights went on or off. There is some evidence that the animals anticipate the dark period; comparison of average numbers of animals above the sand at 1200 and 2000 hrs during the entraining period was significant at the 97.58 level. For the animals in lank 2; during the period of continual darkness, the graphs in Tig. 4 reveal a fairly clear retention of day-night oscillations in activity, coinciding with those of the controls. However. a greater number of animals are at the surface 0900 - 2000 hrs than in the control, and day-night differences between the numbers of animals above the surface at day and those on the surface in the preceeding night, are significant at the 958 level only for May 15 and 17. Reversal of day-night regime periment was designed to test the possibility A seconde of entraining 70 snails to an inverted light:dark cycle - one with the dark period from 0800 to 2000 hrs, Pacific Daylight Saving Time (one hour ahead of PST, beginning 1 May 1972). rocedures were like those just described, except for the reversed light regime. The animals were collected at lower Margaret Stetson low water on May 8 from the sheltered cove on the Boatworks Beach. The results are shown in Fig. 5. The difference between average numbers of animals above the sand in light and dark periods is significant at the 99.998 level for the entrainment period, although there is no obvious consistent anticipation of the light or dark periods. A continuation of oscillations in activity corresponding to the zeitgeber (Aschoff, 1965) is apparent, although the differences between number s above sand during the day and at night is not significant. Additional experiments with numbered individuals showed fairly regular circadian rhythms in several individuals. However, these data were not complete enough to offer an entirely accurate picture of an individual's activity. Aquaria experiments with a simulated tide were set up as well. Data suggests a response to low water levels but, due to insufficient controls and a presence of too many envir- onmental variables, results for these experiments were somewhat irregular. Mating Behavior In attempt to determine whether the number of mating pairs was greater under conditions of light or conditions of dark- ness, control data for the constant darkness experiment (Fig. 4, top) were analyzed. Mean values of pooled data for numbers of mating snails in total light and total dark periods were compared using the Yates correction for Xe. The dif- ference in numbers of mating pairs at night and during the day Margaret Stetson was not significant. DISCUSSION The erratic burrowing behavior of Olivella biplicata and lack of equipment permitting continuoys monitoring of activity make the investigation of the existence of a circadian rhythm 11. in this organism diiricult. In spite of these factors, both field studies and experimental results indicate a definite light:dark periodicity in the activity of Olivella biplicata. Retainment of these oscillationsunder conditions of constant darkness, after being subjected to a light: dark regime, sug- gest the presence of a circadian rhythm. The existence of an endogenous biological clock in Oli vella biplicata, as well as in other intertidal organisms, could be of considerable ecological importance as suggested by J.T. Enright (1970). Although environmental stimuli re- present the dominant determinants of most behavior, an endogen- ous rhythmic pattern could provide the organism with a temporary inertia in times of environmental dis ruptance. the selective advantage of this behavior can be inferred, al- though the behavior of this particular organism appear s to be tremely complex. Summary 1. In the field Olivella biplica ta exhibit a tendency to be above the sand at night and burrowing under the sand in dir- ect light. 2. Experiments on locomotor activity in light and darkness sug- Margaret Stetson gest a correlation between snail activity and presence on the sand surface, both of which are greater in conditions of darkness. 3. After exposure to normal and inverted 12 hr light:12 hr dark regimes under laboratory conditions, populations of Olivella iplicata exhibited oscillations in activity coinciding with those of controls or former oscillations during entrainment, This suggests the existence of a circadian rhythm. Acknowledgements I would like to express my sincere gratitude to Dr. Donald P. Abbott and David Phillips of Hopkins Marine Station for their inexhaustible guidance and encouragement throughout this study. Margaret Stetson Literature Cited Aschoff, Jurgen, ed. 1965. Circadian Clocks, Proceedings of the Feldafing Summer School 7 - 18 Sept. 1964, North-Holland Publishing Co., Amsterdam. Edwards, David Craig 1969. Zonation by Size as an Adaption for Intertidal Life in Olivella biplicata. American Zoologist, 9: 399-417. En ght, J. T. 1970. Ecological Aspects of Endogenous Rhythmicity Annual Review of Ecology and Systematics, vol. 1. Annual Reviews Inc., Palo Alto, Calif. Margaret Stetson Figure Captions Figure 1: Light; dark activity of Olivella biplicata at low tide on sandy beaches. Surf conditions and day:night cycle are illustrated raphically. Black bars represent the number of individuals above the sand and white bars are mating pairs, Time and tidal height at each observation are indicated. Figure 2: Day:night activity of 1000 Olivella on a simulated outdoor beach. Day and night are indicated graphically, with cross hatch being dusk or overcast conditions. Black bars represent numbers of snails on the sand surface. Figure 3: Comparison of locomotor activity of Olivella in light and dark. Top: illustration of tank labyrinth used in the experiment. Bottom: Time after introduction of 25 snails to Box 1 is shown horizontally. The solid line represents the total number of snails above the sand surface at times of ob- servation. Dashed line is the number of snails in Box 1 when observed. The numbers of animals in Boxes 2, 3, and 4 are represented by vertical bars (Box 2- black, Box 3- white, Box 4- cross hatched. Figure 4: Test for a Circadian rhythm in Olivella biplicata. Time and light:dark periods are indicated horizontally. In- dividuals above are indicated by black bars; mating pairs are represented by white bars. Top: control (alternating 12 hour periods of light dark), bottom: experimental tank 12 Margaret Stetson ter 6 days of alternating hour (left in constant darkness a light:dark periods.) Figure 5: Test for entrainment of a circadian rhythm to an inverted light:dark cycle (darkness from 0800 to 2000 hours) Black bars represent numbers of snails on the sand surface, white bars are mating pairs. Light and time are indicated horizontally. 13 — — ...... . â .. . . . â —— — —— . . —.—. ... - — . A281 23 Deer 22 AP8h 2 281 28 Hreu 27 A2820 SVR .... tistt- O 20 —— ..... 2 20 2. S — 4 70 — 1 — 9 6o- - — +I — □ 80- —— 8 Soo .. § 120 â 1—: —0.. 101 .. -GORE + — — .. .... — . L . â â â â ââ â . — . â .. — . — 9 A ro 447 12 So AvH —— — —— ... 300- — — —L S 20ot 00 7 461 7.21 — — 2 — — — EGo — L. — — .... — . —. —.— ———— â .. â . â â —— — . —.— . — — — | — | — —— — — — .. — —— Bor 8 Box Bor —— . .... 20 â 16- . 6164 70 — —— — ) : : T t- —— 2 2 + 1 L — —22- . — —/0 - a —.— L — 2 2 — es.) — —.— Aee . 2 . .. â .... . — — + — :. . 1 ) — — a ——ij—ii-i------------1----——- — dt RiA Mat I A a A77 MA MV8 S30 TANR 1 (oNTol) EE — — — L 62 TANK2 820 10 MR k kaa - LP FlDRE — ———iiiii Mps Mt M4112 a Mat A1 LEEENLT TETTLLE L EEI kk kk kkk- L iL LEE LLRL EEAEEA — FiO —— — .... . ââ âââ 8 +++++n A Mat to Ad Att M17 AA AA12 — — 40 —: 30 â. . — 20 — — 70 L L KA 1E — — —8 S ——— +i Aast8 Hr laMarzo M2M2 MAv23 MA 24 4 — + —.— —— .. .. 30 .... —— —.— —— 20- .. /0 MT T Eigdke . . . . .. . . .