ABSTRACT The range and distribution of the beach isopod Alloniscus perconvexus was found to change over time. Compression and expansion of the range was observed to coincide with spring and neap tides, respectively. The upper limit of the distribution seemed to be set by moisture content of the sand, whereas extreme tidal height appeared to delimit the lower limits of the isopod's range. Range expansion and compression takes place by nighttime activity patterns. While the organism appears to be highly sensitive to the moisture content of the sand, and moves in direct relationship to moisture gradients, its activity can be modified by the presence of light, which inhibits all movement, and by a thigmotactic response which is often enhanced by the presence of potential food. Wave action and physical disturbance may reinforce those factors delineating its lower limits on the beach. DISTRIBUTION OF THE TERRESTRIAL ISOPOD ALLONISCUS PEROONVEXUS ON A SANDY BEACH AND FACTORS AFFECTING ITS RANGE AND POPULATION DENSITI INTRODUCTION The isopod Alloniscus perconvexus (Dana, 1854) has been noted as a common inhabitant of sandy beaches by several authors (Van Name, 1936; Light et al, 1954; and Richardson, 1905). However, little else is known of its biology other than its tolerances to salinity which was examined by Brusca, 1965. The present study was undertaken to determine the distribution of this isopod on a typical sandy beach, and to examine various physical parameters that might affect its range, activity, and population density. Special attention was payed to the influence of moisture content of the sand, tidal height, and the availability of food. Furthermore, laboratory investigations were conducted to determine responses to light, moisture gradients, and to investigate possible thigmotaxis and food preference. MATERIALS AND METHODS Study Area The distribution of Alloniscus was studied on a pocket beach on the southeast side of Point Cabrillo in Pacific Grove, California. A 15-meter wide section of this beach that was relatively uniform in appearance was intensively studied. The beach was semi-exposed, with a slope of approximately .21 meters tidal height. The approximate sand size throughout this section showed a Mdo of -.1. There was a belt of beach grass immediately above the berm, and a large bank of iceplant at the head of the beach. No small rocks were present on the surface and few were buried beneath the sand. Distribution Ten transects approximately 1.5 meters apart were studied under differing conditions of low and high tide, during both night and day. Investigations were made between April 19, 1972 and May 23, 1972. Cores were taken every .75 meters along each transect. The corer (Fig. 1A) was an aluminum box, 25.08 cm x 25.08 cm x 28.57 cm, with two open ends. Four metal strips 24.45 cm x 5.08 cm were attached to the top of the corer to allow the box to be easily withdrawn from the sand. The corer was hammered into the sand, which was subsequently scooped out with a trowel into a sieve with a screen size of 3 mmx 3 mm. At each station, for convenience in handling, the core was divided into two sections. The top 10 cm of sand were sifted first, and the number of A. perconvexus and quantity of algae and large pieces of detritus recorded. The remaining 15 cm of the core was subsequently treated similarly. The sampling along a transect was ended when no Alloniscus had been obtained from two consecutive cores. The distribution of isopods moving on the surface of the sand during the night was likewise studied. Traps were set along a single transect on each of two nights. In one study the traps were set .75 meters apart, and in another, 1.5 meters apart. The traps were glass bowls 5.0 cm in diameter and 10.2 cm deep, positioned in the sand so that the top of the bowl was level with the surface. Organisms walking on the sand fell into the bowls and were unable to climb up the glass sides. Physical Factors To find those factors that might most readily affect the range and population density of Alloniscus, environmental parameters were likewise measured in the sample area. Moisture content of the sand, temperature, presence of food on the surface, presence of objects in the sand, and sand size were monitored for many of the transects studied. When a given tide was at its highest or lowest point, samples of sand were taken every five feet along a single transect, both at the surface and 10 cm below. The moisture content of the sand was calculated by weight loss after drying to constant weight. Temperature was also recorded with standard Centigrade ther- mometers at the sand surface and 10 cm below, every 1.5 meters along the transect. Finally, the presence of grass or detritus on the surface and the quantity of objects in the sand were generally observed and recorded. The sand size was determined by dry sieving through a series of standard Tyler screens according to the methods of Morgans, 1956. Experimental Experiments were conducted on the isopods to test the effects of moisture, light, and the presence of objects and food in the sand. A gradient box made of plexiglas (Fig. 1B), devised by Mr. Samuel Johnson at Hopkins Marine Station, was utilized. Four pieces (42.55 cm x 47.63 cm, 42.55 cm x 47.63 cm, 10.16 cm x 45.08 cm, and 10.16 cm x 42.55 cm) were fastened together to form an open¬ ended box. The two smaller side-pieces were attached so that one was at the same level as the top and the other 2.5 cm below the top. Grooves were cut in the 42.55 cm x 47.63 cm sides, so slats serving as top and bottom could be slid into place (dimensions 45.72 cmx 10.79 cm). Grooves were also cut, perpendicular to the top and bottom, dividing the large sides into four. Three 41.59 cm x 10.48 cm slats could then be slid into these grooves, creating four separate compartments. Moisture Preference The isopods were first tested for moisture preferences. Sand from the study area was completely dried in an oven at 80° C. for approximately 24 hours, and then mixed with sea water to arrive at the desired moisture level. The weight of 1 liters of sand was approximately 733 grams, thus 43.98 ml sea water were added for every 2% increase in moisture content. A gradient of O, 28, 48, and 10 moisture content was set up, using 1 liters of sand per compartment. 10 large Alloniscus (11 mm - 16 mm) were placed in each section and allowed to burrow. The partitions were removed, the sand smoothed, and the top slat slid into place to prevent evaporation. A control box was similarly set up with 2% moisture content throughout. The two boxes were then covered with black plastic sheeting and left overnight at room temperature for 15 hours. At the termination of the experiment, partitions were hammered into the sand. Each compartment was then sifted separately and the isopods recovered. After completion of the experiment, the moisture content of each compartment was determined once again as noted above. Two additional sets of moisture gradients were set up, with O, 48, &, 12, and 10, 12, 1%, 16% moisture content, respectively. Thigmotaxis To measure thigmotactic responses, a gradient of O, 13, 8. and 12% moisture content was again set up, with 4 Macrocystis floats half buried in the sand in both the O and compartments. A moisture gradient containing no algae was used as a control. Salinity - Light Two variations of the moisture gradient O, 28, 48, and 10% were conducted, to test the effects of salinity and light. In one experiment distilled water rather than sea water was utilized to moisten the sand in both experimental and control boxes. In another experiment, using the same gradient, the two boxes were kept under constant illumination from an overhead light. 20 large isopods (11 mm - 16 mm) and 20 small isopods (6 mm - 8 mm) were used per box. Observations were next conducted to test the effects of light and dark on large and small Alloniscus. Four plexiglas boxes were filled with sand of 2% moisture content. 40 isopods were placed in each, two containing large and two with small organisms. Two of the boxes were placed under a bright lamp, and the others outside in the dark. The numbers of isopods remaining on the surface were recorded every hour. Food A test was made to verify the suspected food of A. perconvexus. Dead Orchestoidea californiana and pieces of the algae Macrocystis were soaked in the vital dye Rose Bengal. They were then drained, blotted, and put on the surface of moist sand in separate glass bowls. 10 large isopods which had been kept in the laboratory for two days were introduced in each bowl and placed in the dark. The bodies of the isopods were examined 24 hours later for the presence of dye. A final experiment was conducted to identify possible food preferences. Three plexiglas boxes were filled with 14 liters of sand with 4% water content. In one box, one fourth of the surface of each compartment was covered by a different type of dry wrack: Cystoseira, Gigartina corymbifera, Macrocystis, and Phyllospadix. The second box was similarly filled with damp wrack. The third served as a control, containing no algae. RESULTS Distribution The range and population density changed appreciably during the study as indicated in Figure 2. The transects on April 25 and April 28 were studied after a period of high tides and strong winds; waves had reached tidal heights of 4.8 m during this time. The range of A. perconvexus during these two studies was relatively short, with large numbers of individuals concentrated over a small area. On the other hand, the distributions on May 9, May 17, and May 22 indicated a greater range and a shift in relative numbers. The waves at this time reached tidal heights of only 1.2 to 3.5 m. The weather had been much calmer and the waves had not come so far up the beach previous to these dates. However, on May 27 water came to the 4.86 m tidal height. The range in this transect was once again highly restricted. In contrast, the four distributions shown in Figure 3 indicate that the range of Alloniscus does not change greatly with the four tides occurring during any given 24-hour period. Activity The activity pattern of Alloniscus was also observed on this beach. During the day, the isopod was found burrowed in the sand, a large majority (758) within the top 10 cm of the surface. On dark nights, many came to the top but their behavior could not be observed directly. The isopods stopped movement immediately when illuminated with a beam of white light from a flashlight. Red light produced by taping red cellophane over the flashlight brought about the same response. Traps were found to be the best way to determine nighttime activity. Results shown in Figure 4A were obtained April 22, during the period of higher water described earlier. Note that activity was confined to the upper beach with 32% of the isopods caught between tidal heights 5.7 m to 6.0 m. However, there was a striking contrast between these results and those shown in Figure 4B, taken on May 22 after a period of lower water. No isopods at all were collected between tidal heights 5.7 mto 6.0 m and the isopods were seen to be active down to a tidal height of 3.5 m. An indication of their aversion to light can be obtained from Figure AB. During this study period, the moon was shining brightly until 11:45 PM. Clouds then covered the moon and the surroundings became much darker. Only six isopods were caught before 12:15 AM, whereas 167 in total were obtained from 12:15 AM to 5:15 AM. Similarly, only 3 Alloniscus were caught from 5:15 AM to 6:15 AM, when the sky was gradually lightening. Physical Parameters Sand moisture was next studied along each transect. The surface tended to be consistently dry (below .5% moisture) whereas distinct differences were noted along the transects 10 cm below the surface. In Figure 5, moisture content 10 cm below was plotted against the four distributions taken on May 1, May 17, and the two taken on May 22. The range in which Alloniscus occurs had a water content of .65% to 7.3%. A marked difference was once again evident between the distribution of May 1 and the later four study periods. The range and population density shifted down the beach as the moisture content dropped. On the transect taken on May 1, the percent water at tidal height 4.8 m was 4.34%, whereas the average at this point for the later distributions was only 2.218. Thus, the distribution of Alloniscus appeared to change with fluctuations in moisture content. Note also, that at low tides, Alloniscus appeared in greatest densities in sand of approximately 3% moisture content. Field observations indicated that Alloniscus seemed to congregate in areas in which much algae was buried beneath the surface. As many as 42 organisms were found at a single such location. In Figure 3, the first transect contained a large amount of buried material at 4.5 mtidal height. The next three transects had a scanty amount at this level, and correspondingly fewer Alloniscus were collected. The isopods were found in sand with temperatures 10 cm below ranging from 21.2° C. to 13.2° C. On any single study period, however, there were no drastic fluctuations along the transect, but rather gradual changes (Figure 6). The temperature at the surface showed more drastic changes, with ranges of 27.50 C. to 9.100. Sand size on the surface and 10 cm below did not change drastically along a transect in this section of the beach. Along one transect, from 5.8 m to 3.5 m tidal height, Md o 10 cm below ranged from.4 to.6. Experimental Typical results of gradient experiments designed to determine preferred moisture content showed that Alloniscus congregated to a large extent in the wettest portion (Table 1). Using the chi square test, such a distribution was significant to the 98 confidence level. The average moisture content in the four compartments after 15 hours was .218, 2.93, 5.00%, and 7.42%, while the control was 1.90%, 1.768, 1.90, and 1.878. Similar trends of orientation toward the wettest sand were also found with large isopods tested in moisture gradients of G, 18, 88, and 124 (Table 2). Again the results were significant to the 905 confidence level. The former experiment was duplicated by using 40 small A. perconvexus (6 mm - 8 mm). These smaller animals also moved toward the wettest portion (Table 3). Chi square tests showed this distribution not to be significant, due to an extreme bias in the control. In the controls, the isopods showed a tendency to cluster in one or both of the end compartments. When this data was compared with an idealized control of 10 isopods per compartment, significance to the 95% confidence level was obtained. The control shown in Table 2 also exhibited a large degree of clustering. This was considered to be another indication of thigmotaxis. All of the controls except those run in the light appear to be biased by the tendency of animals to cluster in the corners. Small isopods placed in a gradient of 105, 12%, 14%, and 16% showed little tendency to move (Table 4). While the observation was made that Alloniscus can survive for several hours submerged in sea water, it is suggested that these moisture contents may be well above those normally encountered by the species and activity may be reduced in saturated or near saturated sand. Results from the experiment designed to test for a thigmotactic response (Table 5) showed that more isopods congregated in the 4% sand containing Macrocystis floats than in the control. This distribution was significant to the 99.9 confidence level. Results were drastically different between gradients run in the dark and light. Those run in the dark showed the greatest number of organisms occurred in the sand of highest moisture content (10) and thus was significant to the 99% confidence level. A similar experiment run in the Might, however, showed no significant change in distribution (Table 6). The isopods showed considerable movement in the dark, but much less tendency to move in the light. The boxes in the dark had approximately 60 holes on the surface, indicating that many of the isopods had come to the surface. Those boxes under constant illumination, however, had only 5 holes. Some organisms may have emerged from sand with O moisture content, but the sand was so dry that no trace of any holes was left. In the control run in the light, the typical movement toward one of the end compartments did not occur, while the control in the dark once again showed a a tendency of the animals to aggregate at the corners. The observations made of activity patterns in organisms moving over the surface of the sand were scored every hour in boxes placed in the light and dark are recorded in Figure 7. It is significant that no isopods were seen on the surface in the box kept under constant light, while in the dark, approximately t of those originally placed in the box were observed on the surface every hour. - 12 Food Two types of food of A. perconvexus were found: dead O. californiana and pieces of Macrocystis. The isopods ingested these dyed materials, turning their intestines bright red. The experiment to find food preferences was not successful. The isopods did not tend to cluster in the compartment that held their "preferred" food. Large holes were seen in the pieces of Macrocystis, however. DISCUSSION The distribution of Alloniscus was found to shift in relation to tidal height. The upper limit of the population was quite stable at a tidal height of about 5.6 m, but the lower limit changed drastically with time. The range appears to decrease greatly during spring tides when waves reach the upper portions of the beach. Higher water caused a restriction of the population to the upper beach, while a period of low calm water enabled the range to be expanded to a tidal height as low as 3.6 m during the period of study, It is not known, however, whether this represents the lowest possible range of this species. While distribution of Alloniscus appears to be very much influenced by long term cycles of spring and neap tides, the populations do not move greatly during a single 24 hour period. Moisture content of sand was also believed to play an important role in setting an upper limit for the distribution of these isopods. They were not found at a moisture level lower than .65%. No isopods occurred above 5.6 m tidal height, which was found on one occasion -1 to have a water content as low as.1% and never above.8. Correspondingly, in laboratory experiments, definite movement was observed away from sand with little or no moisture content. Sand moisture content at the isopod's lower limit was less constant, varying from 2.75% to 7.3%. However, the majority of Alloniscus were almost always found in sand with a 28 to 3% moisture content, The presence of detritus and objects in the sand had a great effect on population density, both in the field and in laboratory experiments. Large numbers congregated where many pieces of large damp algae were found buried within the top 10 centimeters. This seemed to indicate either thigmotactic behavior or attraction to a food source. However, it was not possible to determine if the organisms could eat while burrowed in the sand. Sand size was not considered to be a significant factor in determining distribution. Experiments in the laboratory confirmed trends noted in the field but also raised several questions. In moisture gradients, the isopods consistently moved out of the (.218) compartment, in accordance with field data. On the other hand, in the gradient boxes the isopods congregated at a greater moisture level than they were normally associated with on the beach. Apparently other parameters in the field inhibit this movement toward higher water contents. Wave action, physical disturbance, and the presence of detritus might be important variables in this regard. Evidence was obtained to indicate that the isopods were positively thigmotactic. No only did they cluster in areas containing large pieces of algae buried in the sand, but in addition, in many controls run in the laboratory experiments, there was a tendency for the animals to group in the end compartments. While food studies indicated that A. perconvexus was a scavenger, eating surface detritus including wrack and dead Orchestoidea, it was not possible to distinguish between a positive thigmotactic response and attraction to food. Changes in salinity had no effect on the distribution and movements seen in the experiments conducted, and it did not appear as an important factor in the field studies either. Light had a great influence on the isopod's behavior. In the illuminated gradient test, very few individuals came to the surface, and comparatively little movement occurred. Field studies also indicated that Alloniscus were negatively phototactic. They came to the surface only on dark nights; moonlight was sufficient to inhibit their emergence. In summary, the distribution of A. perconvexus was found to change greatly with time. The upper limit of their range was determined by moisture content of the sand, whereas the lower limit seemed to be set by tidal height and the height of the high tides of the spring and neap tides. Population density resulted from movements along the beach. Spring tides caused a compression of the range and a consequent increase in population density in the upper beach. The lower neap tides allowed the range to expand down the beach, in general reducing the density at any given point. Several factors appear to modify this general pattern of distribution. The isopods cluster in areas containing large quantities of buried algae. This is possibly due to a positive thigmotactic response, but may also involve reinforcement through the presence of potential food material. Vertical movement was also observed to be modified, since Alloniscus came to the top of the sand only during the dark. The isopods were very negatively phototactic, and even sensitive to moonlight. ACKNOWLEDGMENTS The author was very grateful to Dr. Welton Lee of Hopkins Marine Station for invaluable assistance during the course of the study and for critical reading of the manuscript. 8 Moisture 06 26 12 104 O 5 6 29 Experimental Control 12 9 7 12 TABLE 1: Moisture gradient run with large Alloniscus. Controls contained sand at 2 moisture content. 10 animals were placed in each compartment. The number remaining was scored after 15 hours in the dark. 2 Moisture OB 16 85 12% Experimental O 3 10 27 Control 11 4 5 20 TABLE 2: Moisture gradient run with large Alloniscus. Controls contained sand at 4% moisture content. 10 animals were placed in each compartment. The number remaining was scored after 15 hours in the dark. 8 Moisture 06 2% 12 105 O 1 8 31 Experimental 3 3 6 28 Control Moisture gradient run with small Alloniscus. TABLE 3: Controls contained sand at 2 moisture content. The 10 animals were placed in each compartment. number remaining was scored after 15 hours in the dark. 2 Moisture 106 12% 118 166 Experimental 9 10 6 9 2 10 9 16 Control TABLE 4: Moisture gradient run with small Alloniscus. Controls contained sand at 12% moisture content. 10 animals were placed in each compartment. The number remaining was scored after 15 hours in the dark. 2 Moisture 05 algae 1% algae 86 124 22 Experimental 18 12 Control TABLE 5: Possible thigmotaxis. Pieces of algae were mixed in the O and 4% compartments in a gradient. Controls contained sand with a , 4%, 8%, and 12% moisture content but no algae. 10 animals were placed in each compartment. The number remaining was scored after 15 hours in the dark. 2 Moisture O 27 16 106 2 11 10 17 Experimental A Light 10 10 10 10 Control O 4 10 26 Experimental B Dark Control 13 10 9 8 TABLE 6: Moisture gradients run in the light and dark. Controls contained sand at 2% moisture content. 10 animals, half large and half small, were placed in each compartment. In 6A, the boxes were placed in the light, whereas they were in constant darkness for 6B. The number remaining was scored after 15 hours. CAPTIONS TO FIGURES Figure lA: Corer used for studies of distribution 1B: Gradient box. Grooves on one side indicated by dotted lines. Distribution of A. perconvexus in numbers per.0156 cubic Figure 2: meters along 6 transects studied on April 25, April 28, May 1, May 9, May 17, and May 28. The intertidal height of the highest waves recorded during each study period is indicated by a horizontal line. The arrow on the last transect (May 28) indicates the height of waves the previous night. Numbers were determined by coring every .75 meters along each transect. The distribution of A. perconvexus in numbers per.0156 Figure 3: cubic meters as observed over a 24-hour period on May 22 to May 23. The intertidal height of the highest waves recorded during each study period is indicated by a horizontal line. Numbers were determined by coring every.75 meters along each transect. Numbers of Alloniscus caught in traps set on May 18 and Figure 4: May 22 from 8:00 PM - 7:00 AM. Collections were taken every hour. 4A: Traps were set every .75 meters along the transect to a tidal height of 5.3 m. AB: Traps were set every 1.5 meters along the transect to a tidal height of 3.2 m. The moon was shining until 11:45 PM, when it was covered by clouds. Figure 5: Moisture content 10 cm below surface and the distribution of Alloniscus determined on May 1, May 17, and May 22. Figure 6: Temperature taken 10 cm below the surface of the sand on the four transects studied on May 22 and May 23. The dotted lines show low tide. The solid lines indicate high tide. Circles show sites where the temperature was taken. Numbers of Alloniscus on the surface of the sand in Figure 7: artificial containers kept in constant light and constant dark. Visual counts were made every hour from 8:30 PM to 6:30 AM. 5 08 cm — 28 57 cm 125 08 cm 25 08cm 10 16 cm L 4763cm --- 42 55 cm APRIL 25 LOW TIDE 2:24 PM APRIL28 MAY I HIGHTIDE LOW TIDE J0:04 PM 7:16 AM KEY — - 10 Individuals MAY 9 HIGHTIDE 8:28 PM MAY17 HIGH TIDE 120PM MAY 28 HIGH TIDE 11O5 PM V — 54 MAY 22 LOW TIDE 1:14 PM KEY — = 5 Individuols MAY 22 HIGHTIDE 8:03 PM MAY 2 LOW TIDE 2:43 AM MAY 2 HIGH TIDE 9:00 AM 9 APRIL18 800 PM - 12:00 AM¬ 7:OO AM 12:00 AM KEY: — =10 hdividuois MAY 22 8:0O PM- 12:0O AM¬ 7:O0 AM 12:00 AM MOONLIGHT NO MOONLIGHT 41 3 6. 6 taatattta- 12 3 4 5 6 7 8 atataaaata- L 5 % MOISTURE CONTENT OF SAND MAYI LOW TIDE 7:16 AM MAY 17 HIGH TIDE 4:20 PM MAY 22 LOWTIDE kI4 PM MAY 22 HIGHTIDE 8:03 PM KEY — 25 Individuals 5 MAY 23 MAY 23 MAY 22 MAY 22 8:03 PM 9:OO AM 2:40 AM 1:14 PM 1 10 11 12 13 14 15 16 17 18 19 20 21 22 23 TEMPERATURE (°C.) 20 10 80 70 60 50 40 30 20 LIGHT 8:30 PM 9:30PM 10:30PM II:3OPM 12:30AM 1:30AM 2:30AM 3:30AM 4:30AM 5:30AM 6:30AN DARK L 8:30 PM 9:30 PM 10-30 PM II:3O PM 12:30AM 1:30 AN 2:30 AM 3:30AM 4:3OAM 5:3OAM 6:30AM REFERENCES CITED Brusca, Gary. 1965. Studies on salinity and humidity tolerences of 5 species of isopods in transition from marine to terrestrial life. Bull. So. Calif. Acad. Sci. 65: 146-154. Light et al. 1964. Intertidal Invertebrates of the Central California Coast. University of California Press, Berkeley, pp. 151, 155, 363. Morgans, J.F.C. 1956. Notes on the analysis of shallow-water soft substrata. J. Anim. Ecol. 25: 367-387. Richardson, H. 1905. A monograph of isopods of North America. Bull. U3 Natl. Mus. 54: 596-597. Van Name, Willard G. 1936. The American land and fresh-water isopod Crustacea. Bull Am Mus. Nat. Hist. 71: 215-217.