ABSTRACT The hermit crabs Pagurus samuelis and Pagurus granosimanis live in close association with the sea anemones Anthopleura xanthogrammica and Anthopleura elegantissima in the intertidal region at Hopkins Marine Station. The crabs are often observed walking on the columns and tentacles of the anemone without any response from the nematocysts. This appears similar in many ways to the relationship established between the tropical anemone fish and their hosts. The hermit crabs go through a sequence of be¬ havior in which they acquire a coating of mucus from contact with the anemone. The mucus is acquired through a series of behavioral steps exhibited by the crabs: poking, brushing, and stroking the anemone. Once acclimated, the crab freely walks among the tentacles feeding off of small food particles stuck to the Anthopleura and even removing food from the gut of its host. The protective mucus coat gradually loses its effectiveness if the hermit crab is isolated from anemones. This is a symbiotic relationship benefiting the crabs by providing a source of food and protection from a potential predator. INTRODUCTION There are many studies on the symbiotic relationship between sea anemones and fish (Lubbock, 1980) (Schlicter, 1976) (Mariscal, 1966) (Davenport and Norris, 1958), anemones and shrimp (Dales, 1966), and anemones and hermit crabs (Hand, 1975) (Ross, 1974). Closely related to these is the curious behavior of the hermit crabs Pagurus samuelis (Stimpson, 1857) and Pagurus granosimanis (Stimpson, 1859) in association with the sea anemones Anthopleura xanthogrammica (Brandt, 1835) and Anthopleura elegantissima (Brandt, 1835). In the inter- tidal of Hopkins Marine Station, Pagurus were observed crawling on the columns, tentacles, and oral discs of the anemones with apparent immunity to their nematocysts. This behavior differs considerably from the hermit crab-anemone association studied by Ross (1974) where the anemone is carried attached to the shell of the hermit crab. These studies were carried out to investigate the nature of the symbiosis chiefly through observing and recording the crabs' behavior during the acclimation process, looking for possible advantages in the relationship and studying the behavior and cooccurrence of the involved species in the field. My studies indicate the hermit crabs acclimate to anemones, perhaps by picking up a coating of anemone mucus. The pagurids may freely crawl over the anemones and take food from the tentacles or even from the gut cavity. The findings presented here indicate a somewhat labile symbiosis between these inter¬ tidal hermit crabs and anemones. MATERIALS AND METHODS Anthopleura xanthogrammica, Anthopleura elegantissima, samuelis, and Pagurus granosimanis were all collected at Hopkins Marine Station on Cabrillo Point, in Pacific Grove, California. All experiments were conducted either in the field or in tanks and aquaria with running seawater. One 40 gallon tank and two 10 gallon aquaria were used for all lab experiments. The 40 gallon tank was located outside, exposed to direct sunlight for approximately 5 hours a day. The aquaria were located in the lab with no direct sunlight. Each tank and aquarium was decorated to simulate a rocky, sandy tide pool. The anemones and crabs were fed a diet of Mytilus, barnacles, and a variety of limpets. All observations at night were conducted with a red light. Hopkins intertidal area evidently offers a very suitable habitat for A. xanthogrammica and A. elegantissima to grow and multiply as there is an abundance of both species. Also present in the same area are Pagurus samuelis and Pagurus granosimanis in great abundance. The research was limited to these two species of hermit crabs and their association with Anthopleura of the size range 5 cm. to 11 cm. Once transferred to the aquaria, the anemones took approxi¬ mately 3 to 5 days to attach and show normal behavior. One noticeable change in behavior was that within a week the stick iness of the tentacles decreased (Mariscal, 1966). For this reason fresh anemones were brought in each week. The hermit crabs taken from the same tidepools in field showed no apparent changes of behavior in lab whereas those taken from separate tidepools exhibited fighting for five days. The crabs from both tidepools were equally as active at night as during the day. Association For a study on the duration of the relation between hermit crabs and anemones, in the field eight tidepools were used, four with anemones and four without. As much care as possible was taken to select tidepools which were similar in exposure, height, and location. In each tidepool shells of crabs were marked with red fingernail polish. For a week, records were kept as to the number of marked crabs remaining in each tidepool. Distribution and Density To determine the distribution and cooccurrence of crabs and anemones in the study area, six transects, 10m x 5m, were established as marked on the map of figure 2. Anemones in the b to 11 cm size range were counted in these transects and hermit crabs were sampled by haphazardly placing .25m quadrats along each transect. This was done for both regions sampled. Acclimation To quantify the shell acclimation of the hermit crabs, I measured the force of adhesion of the tentacles to acclimated and non-acclimated shells using a portable torsion balance that measured up to 24 gms. Tegula were attached by nylon thread and slowly lowered onto the tentacles of A. xanthogrammica and the force required to free the shell was recorded. To test for persistence of the acclimated state 50 empty Tegula shells were acclimated by wiping them on the tentacles of anemones. These were held in an aquarium with running sea water along with 50 unacclimated Tegula shells. Tests were made for the adhesion to anemone tentacles for samples of 5 of each category for the first day at 1 hour intervals after acclimation for 3 hours and then daily for 7 days (refer to Table 1). Haphazard samples of 40 crabs in 3 separate tidepools in the field were tested for acclimation. A simulated tidepool was set up by placing 40 unacclimated crabs into an aquarium with two A. xanthogrammica and after 5½ weeks the number of crabs which were acclimated was recorded. Mucus and Nematocyst Verification In the final experiment to verify mucus accumulation 5 acclimated and 5 non-acclimated shells were placed in flasks with suspended carbon particles. The flasks sat for 24 hours to allow normal settling. Observations were made under a dissecting microscope to detect the presence of mucus. For a verification of nematocyst discharge following crab contact with the anemone, observations were made before and after actual contact with the anemone. Nematocysts were stained using methylene blue dye after scrapings were trans¬ ferred from the chelipeds and shell to cover slips. RESULTS Distribution and Density A comparison of anemone density, hermit crab density, and their association in the study area is shown in Figure 1. West Beach had an average density of 2.7 anemones/m whereas Bird Rock Beach showed 6.2 anemones/m2. These were significantly different by the student t-test (p..05). In the sparsely populated West Beach area 733 of the anemones were A. xantho¬ grammica and only 188 A. xanthogrammica along Bird Rock Beach. The remaining percentages were A. elegantissima. In contrast, the proportion of crabs grazing on the tentacles and columns along West Beach was .25 crabs/anemone as compared to Bird This shows a Rock Beach where there was .5 crabs/anemone. significant difference p..025 (Rx C contingency test). Acclimation Upon first encounter with the sea anemone, the hermit crabs exhibited a behavior I call "poking and retreating." The crabs approached an extended anemone and with their antennae and chelipeds poked at the tips of the anemone's tentacles. Imme¬ diately after each poke they jumped back and retreated a distance. In between pokes the crabs often explored or fed on the immediate area of the rock. This continued for 5 to 30 pokes. Once this behavior was completed, the crabs began a period of brushing their shells on the tentacles of the anemone. Most often the crabs faced away from the anemone, held on to the substrate and placed their shells in contact with the tentacles, They often sat in one spot and moved their shell back and forth through the tentacles. On other occasions crabs would walk back and forth brushing the tentacles. The tentacles often retracted but did not appear to stick to the shell. They displayed this behavior between 5 and 20 minutes. When the brushing was discontinued, the crabs began sweeping and stroking the tentacles with their antennae. The sweeping motion extended along the entire length of the tentacles. An occasional stroke of the antennae would be across the bodies of the crabs. This behavior continued for 5 to 25 minutes, The crabs were then observed freely walking among the tentacles, Not all of the crabs which engaged in some of the steps engaged in them all. However, those that actually walked on the tentacles and therefore were considered completely acclimated did display each step sequentially. Many pagurids completed one or two steps and then left the vicinity of the anemone. The anemone's response to these actions varied. Normally only those tentacles being contacted while being brushed or poked responded with retraction. Within a couple of minutes these tentacles were again fully extended. However, 1 out of 10 anemones responded by slowly and completely closing. It took from 20 minutes to 45 minutes for the anemone to relax and re¬ open. Once acclimation occurred, the only response was an occasional retraction of a tentacle as it was contacted. As the crab walked among the tentacles, it stroked each tentacle in its path with its antennae. Again, as when acclimating, the antennae would occasionally be brought to the body. Some crabs were feeding off of the small particles stuck to the tentacles. On three different occasions, I noticed a crab aggressively entering through the tentacles of an A. xanthogrammica and inserting its chelipeds into the gastric cavity, withdrawing chunks of food and eating. One final ob¬ servation was the crab dislodging detritus which accumulated on the tentacles. Adhesion Tests As seen in Table 1, the acclimated Tegula shells had very little, if any, adhesion to the tentacles. This lasted for several days but adhesion did increase from 1.5 gms. to 7.5 gms. within the week. The unacclimated shells registered an average of 20 gms. or greater with each trial. The results on the number of acclimated crabs after 2 months in the aquarium with anemones showed 32 out of 40 or 803 acclimation as compared to 47 out of 120 or 398 for those sampled in the field. Discharged nematocysts were found on 5 out of 6 of the acclimated crabs. The nematocysts were visible under the compound microscope at 40x power and could be positively identified at 100x magnification. Methylene blue dye colored each nematocyst a bright purple. It was obvious with the dissecting microscope that carbon particles had settled in denser patches on the acclimated shells. The particles appeared supported on a mucus layer that held them above the shell as seen with 45x magnification of the dissecting microscope. The unacclimated shells also accumulated some carbon particles but not nearly as much. All five acclimated shells were easily differentiated by eye inspection from the unacclimated, once the particles had settled. It was also noticed throughout the study that a crab acclimated to A. elegantissima was acclimated to both A. elegan¬ tissima and A. xanthogrammica. However, those acclimated to A. xanthogrammica were not acclimated to A. elegantissima. DISCUSSION These studies indicate intertidal hermit crabs can become acclimated to the large carnivorous anemones that share their habitat. The association is in many ways similar to the many observations that have been made on the clownfish and their host anemones. There are however a significant number of differences. They are similar in that a sequence of behavioral steps is performed in which the symbiote acquires a coating of anemone mucus which is necessary to gain immunity from the host's nematocysts (Mariscal, 1966) (Davenport and Norris, 1958) A recent investigation by (Lubbock, 1980) claims that the immunity is attributable to special properties of the endo¬ genously produced mucus of anemone fish. It differs from the mucus of other fish in being much thicker and contains neutral polysaccharide rather than acidic ones. Hermit crabs produce no external mucus and only lose their stickiness to anemone tentacles after exposure to anemone mucus. The two differ in specificity of association between symbiote and host. The fish establishes onehost and will return in preference to another anemone. The hermit crabs are not closely tied to one anemone and are acclimated to and visit a number of anemones in their range. While the clown¬ fish receives protection from the anemone's tentacles, the crab benefits by feeding from the tentacles and actually with¬ drawing food from the anemone's gut. The crab-anemone asso¬ ciation is essentially a commensal symbiotic relationship benefiting the crab whereas the anemone also benefits from the fish categorizing it as mutualistic symbiosis (Mariscal, 1966). The exposed, steeply graded, rocky West Beach area has a lower density of sea anemones than the protected Bird Rock region and they are primarily A. xanthogrammica. Despite all of these differences this study revealed a remarkable similarity in the pattern of association between the hermit crabs and their host anemones. It is possible that the advantages to the crabs in associating with the anemones compensate for the many environmental differences. This is further supported by the observation of marked stability of residency of crabs in pools with anemones compared to pools without. Another intriguing observation involved the percentage of the hermit crab population in the Bird Rock transect areas which were acclimated to anemones. In an area with an abundance of anemones many clonal ones in addition to the large solitary forms, only 408 of the crabs were acclimated. In the aquarium where the hermit crabs were left in contact with the anemones for over 5 weeks there were still 208 unacclimated. This may result from some intraspecific differences in behavior, social exclusion, stage of life cycle, or simple differences in opportunity. It is certainly an aspect of the study that requires more work. The acclimation behavior pattern of the hermit crabs has some interesting parallels with the acclimation behavior described for the tropical anemone fish. The initial poking and retreating behavior resembles the nibbling of the fish at the base of the column and tentacles. The fish behavior of diving into the tentacles and wriggling through them seems to correspond to the crab's behavior of rubbing the shell on the column and tentacles of the anemone. This probably indicates the most effective behavioral sequence for acclimation to the anemones is strongly conditioned by general properties of the responses of anthozoan cnidoblasts and nerve net. If the initial approach is too abrupt the anemone will be stimulated into either feeding or protective withdrawal behavior and thus terminate the encounter. The poking and retreating initiates contact with the tentacles and may orient the crab to the responses of the anemone and begins habituation of the anemone to the crab, This step probably accumulates mucus on the cheliped surfaces and antennae. The brushing maneuver exposes the shell to the mucus and brings it closer to the entire crab acclimation. Shell acclimation is crucial to the crab for movement through the tentacles without sticking and causing a contraction. Hours of observations suggest that the small appendages and upper abdomen obtain a mucus via the antennae. The observation that unacclimated crabs, if large enough, when placed on the oral disc can escape even after being swallowed by the anemone indicates the crab is well adapted to establish its symbiotic relationship. However, the escape takes quite a bit of time in which the crab could be doing other things. Also when the crabs are too small to free them¬ selves they will be eaten. The acclimation process permits the pagurids to freely exploit their environment, gain another source of food, and perhaps other unrecognized advantages. Where encounters are frequent, as in crowded tide pools, this has obvious advantages for both in simple saving of time. Because of their similarity in times of activity, habitat and population abundance, it seems evolutionarily and eco¬ logically fitting that their daily routines should involve each other. Association may have evolved commensally as the hermit crab acquired a series of behavioral acts to obtain a food source. Benefits if proven for the anemone would give it a more mutualistic definition. ACKNOWLEDGEMENT I would like to thank Dr. D. P. Abbott, Dr. I. A. Abbott, Robin Burnett and Charles Baxter for instruction and counsel during this study. Special thanks goes to Charles Baxter for advice, encouragement, and the editing of this paper. I would also like to thank my mother for coming all the way from Oklahoma to type this paper. LITERATURE CITED Dales, R.P. 1966. Symbiosis in marine organisms. In Henry, Mark S. (Ed.), Symbiosis, Vol. I. Academic Press, New York. p. 478. Davenport, D. 1955. Specificity and behavior in symbiosis. The Quarterly Review of Bio., 30, 29-46. Davenport, D., Norris, K. S. 1958. Observations on the symbiosis of the sea anemone Stoichactis and the pomacentrid fish, Amphiprion percula. Biol. Bull. mar. biol. Lab Woods Hole, 115(3), 397-410. Hand, Cadet 1975. Behavior of some New Zealand sea anemones and their molluscan and Crustacean Hosts. N.Z.J. Mar. Fresh- water Res., 9(4), 509-527. Hazlett, B. 1970. Tactile stimuli in the social behavior of Pagurus bernhardus. Behavior, 36, 20-48. Lubbock, R. 1980. Why are clownfishes not stung by sea anemones? Proc. R. Soc. London, B207, 35-61. Mariscal, R.N. 1966. A field and experimental study of the symbiotic association of fishes and sea anemones. Doctoral Dissertation, Univ. of Cal., Berkeley. Ross, D. M. 1974. Behavior Patterns. In L. Muscatine and H.M. Lenhoff (Ed.), Coelenterate Biology, Academic Press, New York. pp. 281-309. Winston, M. and Jacobsen, S. 1978. Aggressive interactions among hermit crabs. Animal Behavior, 26(1), 184-191. TABLE LEGEND Table 1: Adhesion force exerted by the tentacles of Anthopleus xanthogrammica on empty Tegula shells. Measurements which exceeded 24 gms. maximum are recorded as 24+. FIGURE LEGEND Figure 1: Distribution map and graph showing the densities of Anthopleura xanthogrammica and Anthopleura elegantissima, the densities of hermit crabs and the numbers of crabs associated with anemones for 6 transects around Cabrillo Point at Hopkins Marine Station. Transect 1 and 2 are considered West Beach transects. 3, 4, 5, and 6 are con¬ sidered Bird Rock Beach transects. Figure 2: Residency of marked crabs in pools with and pools without anemones. Table 1 hour .5 1.5 2.5 day Adhesive force grams Acclimated Non-acclimated range mean range mean 10.0-244 22.04 0-4.0 7.5-24+ 13.04 1.5 0-2.0 20.0-24+ 1.0 24.0+ 0-3.0 2.0 18.0-24+ 20.0+ 1.0-6.0 4.0 20.0-24+ 22.0+ 0-3.0 21.0+ 1.0 12.0-24+ 24+ 0-3.5 24+ 1.5 4.0 24+ 24+ 0-5.0 24+ 24+ 4.0 0-6.0 24 + 1.0-4.0 24+ 2.0 24 + 24 + 2.0-9.5 7.5 24 + 2.0-10.0 24 + 6.5 1.0-12.0 7.5 24 + 24 + 16 9 28 25 u 5 E+ 2 0- 9 58 EOE 10 18 18 Figure 2 50 40 30 20 10 F 50) 40 30 20 10 WITH ANEMONES Number of Days WITHOUT ANEMONES O -8= Number of Days -0 O -— 5 9- 18