Determinants of Activit in A. perconvexu NTRODUCTION It has been theorized that the terrestrial isopod crustaceans of the suborder Oniscoidea represent forms which have migrated from the ocean and have thus acquired terrestrial habitats (Miller, 1938). They may represent a transitional stage in the evolutionary movement of fauna from the sea onto the land. There is considerable variance between the different Oniscoidean species and the degree of their adaptation to the land environment. One species, Alloniscus perconvexus, occupies the upper littoral zones of the sandy beach, above the high tide level (Ricketts and Calvin, 1968). It is generally burrowed in the sand at a depth of 7 to 12 cm (Brusca, 1966). Its distribution on the beach is limited below by the high tide level and above by the degree of moisture in the sand (Hainsworth, 1972). Water relations are of the greatest importance in determining the local distribution of isopods on land (Miller, 1938). A. perconvexus is not adapted to complete submergence and will drown in seawater (Ricketts and Calvin, 1968). A. perconvexus respires primarily through its gill surfaces, though some special air chambers have been noted at the edges of the exopodites which may function as accessory respiratory organs (Miller, 1938). The gill surfaces must be kept moist if they are to function effectively. A. perconvexus consequently requires a habitat of greater moisture content than Porcellio scaber and other terrestrial Oniscoideans. This shows a lesser degree of adaptation to the land Determinants of Activity in A. perconvexus environment. Observations of the activity of A. perconvexus by this author have suggested that it leaves its daytime burrows at night to actively move about on the sand surface. The animal is thus behaviorally protected from the desiccating influences of the lower daytime humidities by remaining in burrows during the daylight hours. The determinant factors for the activity of A. perconvexus may be either exogenous or endogenous. The stimulus for activity could be an environmental cue or may be related to the internal physiology of the animal. Studies on the terrestrial isopod Oniscus asellus have shown that its endogenous diurnal activity patterns are correlated with the alternating light-dark cycle, and not with fluctuations in temperature and humidity (Cloudsley-Thompson, 1961). The light-dark cycle is a more regular cycle for an organism to be synchronized with. An endogenous rhythm would be a useful adaptation for an isopod which might have no other means to obtain information about external conditions which might themselves be acting as stimuli for its activity patterns (Enright, 1970). Such a mechanism would preclude activity before conditions were favorable. This would substancially benefit A. perconvexus. The present experiments were conducted from April to June, 1973. with the objective of ellucidating the major factors which determine the activity patterns of the sand beach isopod, Alloniscus perconvexus. Determinants of Activity in A. perconvexus METHODS This investigation was based on field observations and laboratory studies. The population of Alloniscus perconvexus studied was found on the upper portion of the northeast facing sandy beach between Pt. Cabrillo and Pt. Alones on Monterey Bay, California. Field Studies Field studies of the activity patterns of A. perconvexus were conducted using 26 pitfall traps consisting of plastic cups (diameter of 8 cm) buried in the sand and flush with the surface. These traps were placed 0.5 to 1.0 m apart, and 5 cm from any rocks or deposits of wrack. The study region was 7 m square. Traps were examined at hourly intervals at night during April and May. Collected animals were immediately released at least 1.5 m from any trap, and those caught in any one trap were not all released in the same place. This reduced the probability of catching the animals consistently in the same trap while still keeping them in the same local area. Initially a 24 hour observation period was conducted to establish the overall pattern of activity in the field. This was followed by observations during the active night periods on 3 successive evenings, during which the environmental factors of air temperature and relative humidity weremonitored at hourly intervals. Temperature was measured with a tele-thermometer and humidity with a Honeywell relative humidity indicator. Wind speed was negligible on all 3 nights and was not recorded. A second 24 hour observation was conducted i month later Determinants of Activity in A. perconvexus to ensure that no change in activity patterns occurred during the course of the studies. Laboratory Experiments Laboratory experiments were conducted at the Hopkins Marine Station in Pacific Grove, California. Phototropism was studied by applying slight modifications to the procedure and apparatus described by C. H. Abbott (Abbott, 1918) in his studies of the responses of Porcellio scaber to light. The light source consisted of an enclosed box with a slit opening and a high intensity light bulb (connected to an American Optical voltage selector). Water in a rectangular container was a coolant for the light beam passing through the slit (see details in Appendix A). In this set of experiments, a circular experimental grid was laid out on a board covered with brown paper. This surface precludes burrowing by the animal. The light source box was then placed on this board and the entire apparatus was placed in a darkroom of 14°0, which is slightly warmer than the nightly air temperature. Individuals were tested by placing them in the center of the experimental grid and noting where they left the grid. A positive response consisted of a any individual leaving the circle in the quadrant which faced the light. An individual was tested in each-of 4 orientations relative to the direction of the light (see Appendix A). Four runs were made. The first run used 9 individuals collected at 0100 and tested after a period of acclimation to the experimental conditions. The second run used animals collected at 1600 and tested at that time. The leterminants of Activity in A. perconvexus third run used animals collected the previous day at 1400, acclimated for 24 hours, and tested at 1400. The final run used the animals of the previous test, only they were tested 12 hours later at 0200. Controls consisted of similar runs conducted with no light source. A second set of phototropism experiments was conducted using a sand surface which allowed the animals to burrow normally. A bottom- less box 37x7.5x10 cm, open at both ends, was placed into a tub containing sand from the natural habitat. This comprised the experi¬ mental chamber. The sides were imbedded 3 cm into the sand. One half of the chamber had been darkened inside by black paper, and another sheet of black paper was placed over that open end. The other end was oriented towards a dim light of 0.14 ft candle intensity, measured by a Photovolt light meter. A pitfall trap was placed at either end, and 20 animals were introduced in the middle of the chamber. After 2 hours the traps were emptied, and the process was repeated for 20 more animals. These experiments were conducted at night in the 1400 darkroom. A third set of phototropism experiments was conducted in the 14°0 darkroom to determine a possible threshold light intensity at which the animals might leave their daytime microhabitats. The experi¬ mental chamber consisted of a box (4OxiOx10 cm) with one half open on top and the other half completely enclosed as a dark chamber, and only a small 2.5x2.5 cm opening between the two halves. The same high intensity light mentioned earlier was placed 5 cm above the end of the open chamber. Fifteen A. perconvexus were collected at 2030, in A. perconvexus Determinants of Activit shortly before their natural activity would begin, and placed into the dark chamber for a short acclimation period. The light was turned on and the opening between the two chambers was uncovered. The number of animals seen in the lighted chamber was noted after 5 and 10 minute intervals, and the procedure was repeated at decreasing light intensities. The experiment was repeated the next evening with different animals. Experiments to study the effects of relative humidity on the burrowing response of A. perconvexus were conducted in covered humidity chambers consisting of glass dishes containing 2.5 cm of sand. Small crucibles containing distilled water and calcium chloride desiccant were used to produce relative humidities of 100% and 50% respectively, The chambers were placed in the 14°0 darkroom. Ten animals, collected at night, were placed in each chamber after a short acclimation period. The covered chambers were examined after 30 and 60 minute intervals to determine the number of burrowed isopods. Studies to detail activity patterns were conducted on a laboratory population of 50 individuals established in constant darkness under constant conditions. The population was maintained in an open plastic tub measuring 30x17x10 cm and containing 5 cm of sand. Phyllospadix wrack was used as a food source, and was replaced periodically as it dried out. Small amounts of water were added as needed when the sand became dry. The isopods for the laboratory population were collected from beneath rocks on the beach on the afternoon of May 7, and were placed Determinants of Activity in A. perconvexus into the constant darkness conditions at this time. Observation of activity patterns began on May 9, the criterion for activity again being presence on the sand surface. Activity was measured by visual counting with a flashlight masked with several layers of red cellophane. The observation period lasted a maximum of 15 seconds, and was repeated at intervals of i to 2 hours throughout the active period. These observations were continued for a 3 week period, and totalled i1 obser- vation periods. A second population of 50 A. perconvexus was established in laboratory conditions identical to those of the previous population. This population was maintained undisturbed in constant darkness for a period of 6 days. After 6 days the population was placed on a reversed light-dark cycle for a period of 7 days. Illumination was provided by a standard 60 W light bulb which was turned on at 2000 and off at 0800, this representing an approximate 12 hour shift in the light-dark cycle. The light was turned off at 0800 on May 31 after 7 days, and the population was kept in constant darkness for the duration of the research period. Activity levels were monitored by the same means used in the previous experiment. A control experiment to evaluate the degree of disturbance due both to the flashlight was conducted concurrently with of the previous experiments. Five individuals were collected at night and placed in separate glass containers, each containing 2.5 cm of sand and some Phyllospadix wrack. These dishes were placed under conditions of constant darkness and were undisturbed by flashlight for a 6 day Determinants of Activity in A. perconvexus period. The individuals were then observed periodically at intervals of 3 or 4 hours for one day to check activity levels at different times. A second control experiment was conducted to check the possi¬ bility of light leaks in the darkened chambers containing the 2 laboratory populations. Strips of Kodac photography paper were introduced into the chamber and removed after periods of exposure of 1, 2, and 60 minutes. The paper was then developed to see if it had been exposed by light. RESULTS The field population studied showed a clearly delimited period of activity during the night hours (Figures 1, 2, and 3). Peak activity generally occurred between 2200 and 0200. Little correlation was noted between environmental factors and the initiation of activity (Figure 3). There is no consistent relation between fluctuations in activity and corresponding fluctua¬ tions in temperature or humidity. There is a suggestion of a relation between activity and saturation defecit, as no activity is seen when the saturation defecit exceeds 2.5. A strong correlation existed between the onset of activity and sunset, and between the cessation of activity and sunrise. A. perconvexus shows a statistically significant (p.O1 in a chi square test) positive phototropic response in all cases where the experiments were conducted during night hours (Figure 4). In Determinants of Activity in A. perconvexus addition, a significant photopositive response was observed during daytime tests after the animals had been acclimated for 24 hours in the darkroom (p.Ol in a chi square test. Animals taken from the environment and tested immediately show a random response to light (p=.05 in a chi square test). The controls of May 17 and 18 show no significant responses when the light was off (p.9 and p.25 respectively in a chi square test). The second phototropism experiment (Figure 5) also indicates a photopositive response (p .01 in a chi square test) while the unlighted control indicates a random response (p.25 in a chi square test). As the wide 95% confidence intervals indicate, the sample size in the light intensity threshold experiment was quite small (Figure 6). Although a trend can be seen in the generally increasing responses of the animals towards light as the intensity decreased. no statistically significant threshold can be established for the response. Significant differences are seen after 5 minutes between the response at 0.02 ft. candle and 0.2 ft. candle, and after 10 minutes between the response at 0.02 ft. candle and 0.1 ft. candle. It appeared that animals started responding positively to light at levels below 0.1 ft. candle, though this can't be verified statis¬ tically with this sample size. A significant correlation between relative humidity and burrowing was clearly evident (Figure 7). The animals burrow significantly more at 50% than at 100, as the 95% confidence intervals indicate. The response is more pronounced after 60 minutes exposure to the terminants of Activity in A. perconvexus humidity level than after 30 minutes. The population kept under constant dark conditions exhibited no significant difference in activity pattern from the field population, even after 3 weeks (Figure 8). The peak of activity fell within the same general range of 2200 to 0200. Although occasional individuals were observed to be active at times earlier or later than those observed in the field, it was not a consistent or common occurrence. Slight irregular changes in the amplitude of activity were observed. A. perconvexus retained its 24 hour activity pattern throughout the entire 3 week period. The second laboratory population acquired a new activity pattern, quite different from that observed in the field, during the 7 day period of entrainment to the reversed light regime. After the first period in constant darkness, both the overall period of activity and the time of peak activity shifted forward 2 to 3 hours each period on successive days until the peak activity was once again near 2400, where it remained fixed (Figure 9). After 7 days of entrainment to the reversed light regime, the activity pattern of the population shifted to that associated with a normal light regime after 4 periods in constant darkness. This pattern corresponds to that observed both in the field and in the other laboratory population. DISCUSSION The fundamental issue examined in this study is whether the leterminants of Activity in A. perconvexus activity pattern observed in A. perconvexus is a response to exogenous environmental cues or is due to endogenous influences. A definite pattern of nocturnal activity is exhibited by A. perconvexus (Figures 1, 2, and 3). This is a useful adaptation in view of the animal's moisture requirements. The animal is less exposed to desiccation since relative humidities are greater at night with the lower temperatures. However there is no correlation between activity and fluctuations in temperature and humidity (Figure 3). A. perconvexus was never observed active when the saturation defecit exceeded 2.5. The threat of desiccation becomes greater as saturation defecit increases. No significant correlation between activity and the saturation defecit was noted. As data for day III in Figure 3 indicate, activity had generally ceased by 0600 although the saturation defecit was still a very favorable 0.10. Temperature and humidity are unreliable cues for activity because their daily cycles are irregular. Saturation defecit, being a function of both temperature and humidity, might not be a reliable stimulus either. These factors can thus be eliminated as stimuli for the initiation and cessation of activity. Field observations suggest that light is the stimulus to which the animal responds. The animal was never seen active before sunset. and only isolated individuals were detected after sunrise. Light would be a very reliable stimulus for A. perconvexus to respond to because of the regularity of the daily light cycle. The nightly increases in relative humidity and decreases in terminants of Activity in A. perconvexus temperature and saturation defecit appear to be integrally related to the light-dark cycle in establishing the activity pattern of A. perconvexus. The animal profits from the nightly moisture conditions while it also has a reliable stimulus to define its active periods. The nature of the effect of light on A. perconvexus is uncertain, for the animals may be responding to light directly as an exogenous stimulus, or light may be the entraining agent for an endogenous rhythm. The definite photopositive response of A. perconvexus (Figures 5 and 6) is seen only in those animals which had previously been acclimated to the 14°0 darkroom. Those tested directly from the field exhibited a random response. Though statistically insignifi- cant, there was an apparent trend towards an increased positive response to light at lower light levels (Figure 6). These responses could indicate that A. perconvexus is responding to low light levels as a stimulus for its emergence at night. This could, however, be a response to some cue besides light. The relative humidity in the darkroom where the experiments were conducted was 60%, with a saturation defecit of 4.8. This defecit is considerably higher than the maximum level of 2.5 seen during field activity. Cloudsley-Thompson (Cloudsley-Thompson, 1958) found that Porcellio scaber becomes photopositive when in danger of desiccation. This response allows them to relieve the moisture stress by moving towards a more favorable habitat. A. perconvexus could conceivably be exhibiting the same behavior in leterminants of Activity in A. perconvexus the dry 14°0 darkroom. A relative humidity of 50% was sufficient stimulus to induce burrowing (Figure 7), presumably to avoid desiccation. The satura¬ tion defecit at 14°0 and 50% humidity is 6.0, which is slightly greater than that at 60%. Animals at 14° and 60 humidity may be facing a similar desiccation threat, and respond to the light positively as P. scaber was seen to do. This is consistent with the observation that several animals which had demonstrated a random or photonegative response when first tested would exhibit a definite photopositive response after acclimation in the darkroom. It would be an adaptive advantage for A. perconvexus to show a photopositive response under desiccation stress when burrowed in the sand. To move to a more favorable microhabitat through the sand would be a difficult process, and the animal would be exposed to the desiccating influences for a greater period of time than it would if it moved along the sand surface, even though it would then be exposed to light. The animal can move more quickly along the sand surface, and would be exposed to desiccation for a shorter period in moving to an area of greater moisture content. This behavioral adaptation appears to explain the nature of the observed photo- positive responses in the laboratory. The retention of the same 24 hour periodicity in activity in a laboratory population of A. perconvexus as seen in the field is not sufficient evidence for the presence of an endogenous rhythm. though it may be suggestive of one, even after a 3 week period. Jeterminants of Activity in A. perconvexus Some unknown factor in the environment may be effectively acting as a stimulus to which the animals are responding. Light couldn't be the factor because the only light received was from brief exposure to the masked flashlight at regular intervals. As the 5 control animals indicated, the flashlight apparently had no influence on their activity; they followed the same 24 hour periodicity though they were undisturbed for 6 days. The controls further indicated that individual isopods become active independent of other isopods. Activity isn't initiated by a single individual. No evidence of a spontaneous frequency, or free running period as defined by Pittendrigh (Aschoff, 1960), is seen, other than the 24 hour period observed in A. perconvexus. Seven periods of obser- vation under constant conditions are required before spontaneous frequency can be mentioned relative to an animal's activity pattern (Aschoff, 1960). A. perconvexus has followed the same periodicity in its activity for a 3 week period in constant conditions. This periodicity is in synchrony with the 24 hour light-dark cycle. This suggests that A. perconvexus has a spontaneous frequency of 24 hours. After a 7 day entrainment period to a reversed light-dark cycle, the population exhibits the expected acquired activity pattern, which is in synchrony with the reversed light regime. on the first day in constant darkness. The activity pattern then gradually shifts forward each day until it becomes fixed on the pattern already seen in the laboratory and in the field. The dark eterminants of Activity in A. perconvexus chamber was effectively shielded from light, as the test with the photographic paper indicated (none had been exposed by light after different periods in the chamber). The brief exposure to the masked flashlight in the earlier control seemed to have no effect on the animals. Light cannot be the stimulus for the observed change in the activity pattern after entrainment. The entrainment process indicated that light is a crucial determinant of activity in A. perconvexus. No activity was observed during the lighted periods of the reversed light cycle, although this was the period of normal field activity. The animal was responding to the light in the laboratory, as other conditions remained constant. The 7 day artificial light stimulus wasn't sufficiently powerful to permanently entrain A. perconvexus, for once it was removed some stronger stimulus acted to shift the activity pattern forward to again be synchronized with the normal light-dark cycle. If this stimulus were some exogenous cue, a sudden switch in the pattern might be expected, yet here a gradual change was observed. There is strong evidence for the presence of an endogenous rhythm which determines the activity patterns of A. perconvexus. This is a powerful rhythm and is in synchrony with the periodic fluctuations in the light-dark cycle. It would appear that the transitions between light and darkness are the important natural factors which maintain this rhythm. This would explain the failure of the artificial light stimulus to permanently entrain the animals Determinants of Activity in A. perconvexus to a different light regime, for there were no simulated periods of dusk and dawn. It may also be true that the duration of the artificial light stimulus (7 days) was too short to permanently entrain them. This seems unlikely, however, particularly since the animals were in constant darkness for 6 days prior to the intro introduction of the reversed light regime. Endogenous factors appear to be the primary stimuli determining the activity patterns of A. perconvexus. Light can overcome the endogenous factors to become the primary stimulus under the artificial conditions seen here in the laboratory. The influence of the light is lost quickly once the animal is again in constant darkness. The more powerful endogenous rhythm overcomes the effect of light in determining activity, and the natural activity cycle of the animal is restored. Determinants of Activity in A. perconvexus SUMMARY 1. Field studies have indicated that Alloniscus perconvexus, the sand beach isopod, exhibits a definite pattern of nocturnal activity, with peak activity between 2200 and 0200. 2. Laboratory studies indicated that A. perconvexus demonstrates a positive phototropic response when threatened by desiccation. 3. Strong evidence was found for the existence of an endogenous rhythm in A. perconvexus which is in synchrony with the natural light-dark cycle. This rhythm seems to determine the activity patterns of the animal. This is a powerful rhythm, for after 7 days of entrainment to a reversed light regime, the activity pattern of a population of A. perconvexus shifted within 3 days to that pattern associated with a normal light cycle once the population was placed into constant darkness. LITERATURE CITED Abbott, C. H. 1918. Reactions of Land Isopods to Light. J. Exp. Zoo. 27:193-246. Aschoff, J. 1960. Exogenous and Endogenous Components in Circadian Rhythms. Cold Springs Harbor Symposia on Quantitative Biology. XXV:11-29. Brusca, G. 1965 Studies on Salinity and Humidity Tolerances of Five Species of Isopods in Transition from Harine to Terrestrial Life. Bull. So. Calif. Acad. Sci. 65:146-131. Cloudsley-Thompson, J. L. 1958. Woodlice, p.1-14. In J. L. Cloudsley-Thompson, Spiders, Scorpions, Centipedes, and Mites. Pergman Press, New York. Cloudsley-Thompson, J. L. 1961. Rhythmic Activity in Animal Physiology and Behavior. Academic Press, New York, 261 p. Enright, J. T. 1970. Ecological Aspects of Endogenous Rhythmicity. Ann. Rev. Ecol. Sys, i:221-229. Hainsworth, B. 1972. Personal Communication. Miller, M. A. 1938. Comparative Ecological Studies on the Terrestrial Isopod Crustacea of the San Francisco Bay Region. Univ. California Publ. Zool., 43(7):113-142. Ricketts, E. F., and J. Calvin. 1968. Between Pacific Tides. Ath ed. (J. W. Hedgepeth, rev.), Stanford Univ. Press, 641p. FIGURE LEGEND Figure 1. Pattern of activity for A. perconvexus in the field on April 26. Figure 2. Pattern of activity for A, perconvexus in the field on May 28. Figure 3. Patterns of activity for A. perconvexus in the field (lower series of graphs) with corresponding environmental factors (upper'series of graphs. Environmental factors of temperature (—), relative humidity (—--), and saturation defecit (—:) are represented. The scales for the vertical axes on the upper graphs are temperature in degrees Celsius (left vertical, large numbers), relative humidity in percent (left vertical, small numbers), and saturation defecit (right vertical). The successive nights under observation were: April 30 (I), May 1 (II), and May 2 (III). Figure 4. Phototropic response in A. perconvexus. Vertical axes represent the number of responses. Figure 5. Phototropic response in A. perconvexus. Figure 6. Percent positive responses to light in A. perconvexus at varying light intensities. Included are the 95% confidence intervals. Figure 7. Burrowing response in A. perconvexus at 2 different relative humidities. 50%-Low, 100%-High. Figure 8. Activity pattern seen in a laboratory population of A. perconvexus kept in constant darkness for a period of 3 weeks. Periods of no observation (—) Figure 9. Activity pattern seen in a laboratory population of A. perconvexus kept in darkness after a 7 day period of entrainment to a reversed light regime, light received from 2000 to 0800 during that period. ACKNONLEDGEMENTS I would like to thank Dr. Malvern Gilmartin, without whose help and guidance, particularly in the preparation of the manuscript. this paper would not have been possible. Special thanks are due to my colleague Robert Barmeyer, whose ideas and suggestions were invaluable to me throughout this investigation, and also to Raymond Douglas Davies, Ken Loggins, Jim Messina, and Karen Ransom for the inspiration they provided me in this project. FIGURE 1 — 1 1230 0030 1830 1230 0630 TIME iLll —— ——.—.— —— — | —| — APRIL 26-27 — — — — — — — — - —— 100- — 90 — 80- — —— — | — — — —— 1 — —— — — 70- —— — — — — 1:. 50. — — 40 30 - —— — — — H L — —— —— + — — — —— —tatatt — — — — . — . L --- - —— FIGURE 2. t o ---— TIME 101 MAY 28-29 —— — ———:- - - ——60 — 50 — — —.— — 1 30 7 + 10 1 — — —— — — 10 — — — k + — — — — —— — — — .. — . — — +— —:— — + + — —— — t — —:— — — — - — 1 — O —— —.1— — — CV. OO FIGURE 3. —— — —— - —— — —— 1-. — ——— + - —.— -0630 —— — .-7— — —-.- — — —. — — -0030 L â — —— — + 1830: .. + — - — 1. 0630 — ... i. 0030 — —1— — — 1830: -0630- — — —— 0030 1 — —— 1830 JAILOV ON .. .... FIGURE 4 — —.— 40 0.40 FT. CANDLE ILLUMINATION —— 30 —1: — — —.— 20 10 + — — —— 0200 160C POSITIVE RESPONSE — — MAY II MAY 16 + MEGATIVE RESPONSE LE ——— 1 — — 0,40 FT. CANDLE-ILLUMINATION— 7— 66 . 1 —+ X —4 S SO 1 30 30 CONTROL-NO ILLUMINATION — — — —— 0 20. 10 O200 1400 MAY 18 MAYI7 + EE 56 O200 1400 — — 0.04 FT. 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EXPOSURE EXPOSURE —— I 1. — —— — — —— — — FIGURE 8. . . — + . â — —:—— .. 1 + : — — ——— 1— 1 1 —:— — —+ O ————— .—— V +:—— — — — Ovô —.— — — —.—+ — — — —.— O1 ——— -1 -—:— — + 1 ——— OOvô — — —— —-— —— . 1 - —.— . 2 1—. oët — — ++ —.— — — —— —— — — .—.— 1: —— 4- - OOvô — 4 — —— 2 — + — — — 5 OOët ——.— — — V - — + -!— ----- — — -- - --. OOt . —.— — A —I. ..... el- — 0 —--- JAIIOV ON - - --- FIGURE9. 1— — —— — 1 .— 11 — +—.- 1 V 1- — 0090 — + —:—----- —.— —1— — .— — — — —— — — 0081 — 1 ——- —1—+ —+ —: —— 0090 —.— — — — — — 4 —— 0081. —1 — 7 — — — — 9090 — — — 7 —.7—--—-- 4 8 S 200 8 — — 2 — = V . - — 2 0090 5 —+ — 1 — — 2 X 1 1 — —.— .--. — ) A 5 —1 .. X -— —+ — — 7 12 — 1. + —— — —0090— --—— JAIIOV-ON — —— —— . . 11 APPENDIX A EXPERIMENTAL GRID LIGHT SOURCE BOX 1 4cm; 19.5cm + 10cm—19.5cm- -28cm 12.5cm O.3em. + WATER COOLANT + 0.5cm¬ DIRECTION OF ILLUMINATION EXPERIMENTAL GRII ORIENTATION OF EACH ANIMAL CENTER OF GRID PLACE ANIMAL HERE ANEGATIVE RESPONSE POSITIVE RESPONSE