BULLETIN OF MARINE SCIENCE. 30(2): 460-466. 1980 CORAL REEF PAPER

ACCLIMATION OF TWO SHRIMPS OF THE GENUS TO SEA ANEMONES

Daniel M. Levine and Orland J. Blanchard, Jr.

ABSTRACT Through an acclimation behavior, Peric/imenes rathbunae Schmitt and Periclimenes an- thaphi/us Holthuis and Eibl-Eibesfeldt acquire protection from the tentacles of their respec- tive host sea anemones, Staichactis helianthus (Ellis) and Candy/actis gigantea (Weinland). Laboratory experiments have shown that after a period of isolation from the host these shrimps lose their protection from nematocysts. Unacclimated (=unprotected) shrimps were stung by anemones with and without shrimps living with them, while acclimated (=protected) shrimps were not stung by anemones, with or without shrimps.

Many shrimp belonging to the family are found in sym- biotic association with other invertebrates, including actinians (Chace, 1958, 1972; Mahnken, 1972; Sargent and Wagenbach, 1975; Herrnkind, Stanton, and Conklin, 1976), scyphozoans (Bruce, 1972a), zoantharians (Bruce, 1973), antipatharians (Davis and Cohen, 1968), anthozoans (Bruce, 1972b), echinoids (Bruce, 1972c; Castro, 1974), and molluscs (Shoup, 1972; Bruce, 1975). In the shrimp genus Periclimenes some species associate with sea anemones while others, in addition to living with sea anemones, are cleaning symbionts of reef fishes (Chace, 1958; Limbaugh, Pederson, and Chace, 1961; Feder, 1966; Mahnken, 1972; Sargent and Wagenbach, 1975). Several of these invertebrate hosts, most notably sea anem- Ones, possess stinging nematocysts that are utilized in prey capture, but their shrimp symbionts are apparently not affected by them (Feder, 1966; Mahnken, 1972; Herrnkind et al. 1976). It is clear that the exoskeleton of the shrimps per se do not protect them from the nematocysts of sea anemones. We have seen non-commensal stung by the sea anemone Stoichactis helianthus (Ellis), and Herrnkind et al. (1976) observed that large non-commensal crustaceans were stung by the sea anemone Lebrunia danae (Duchassaing and Michelotti). A similar symbiotic relationship exists between certain pomacentrid fishes and sea anemones (Mariscal, 1972; Allen, 1975). Early workers who studied the po- macentrid fish-anemone symbiosis discovered that the fish became vulnerable to the nematocysts after a period of isolation from their host and had to undergo a behavioral process known as acclimation (Davenport and Norris, 1958; Mariscal, 1965, 1966, 1969, 1970a, 1970c, 1971; Schlichter, 1967, 1968). These workers concluded that a change occurred in the mucus coat of the fish that provided protection from the nematocysts. Schlichter (1970, 1972) performed a series of experiments in which he used sea anemones labeled with tritiated amino acids. The tritiated label was taken up by the sea anemones and accumulated in the surface mucus of the tentacles. Previously isolated anemonefishes were then al- lowed to acclimate to the anemones. Through the use of autoradiographic tech- nique, Schlichter (1972) found that the tritiated label was transferred onto the surface of the fish during acclimation. The highest concentrations of the label were found in those areas which most frequently contacted the tentacles. Schli- chter (1972) concluded that the fish were "chemically camouflaged" as a result of incorporating the anemone mucus into their mucus coat. It is not known wheth- er the anemone mucus forms a complex with the mucus coat of the fish or whether it masks it in some way. 460 LEVINE AND BLANCHARD: ACCLIMATION OF SHRIMPS TO SEA ANEMONES 461

Table I. Results of isolation experiments for Periclimenes rathbllnae and its anemone host, Sto- ichactis helianthlls

Anemone Anemone Without With Total No. of Shrimp Shrimp Shrimp Observ~tions Summary of Results

U naccl imated Unacclimated + + (10) (10) 20 shrimps stung Acclimated Acclimated (10) (6) 16 shrimps not stung Total no. of observations 20 16 36

Numbers in parentheses refer to number of experiments + = shrimp slung; - = shrimp not stung.

The purpose of this study was to determine through a series of laboratory behavioral experiments the protective mechanisms involved in the symbioses between the shrimp Periclimenes rathbunae Schmitt and the sea anemone Sto- ichactis helianthus (Ellis), and the shrimp Periclimenes anthophilus Hothuis and Eibl-Eibesfeldt and the sea anemone (Weinland). The ex- perimental design was patterned after Mariscal (1969, 1970a, 1971) in studying the pomacentrid fish-anemone symbiosis. Reciprocal experiments were conduct- ed in which both acclimated and unacclimated shrimps were tested against anem- ones with and without (acclimated and unacclimated, respectively) shrimps living with them. The rationale behind these experiments was as follows: if only un- acclimated shrimps were stung by sea anemones, both with and without shrimps, this would indicate that some change had occurred in the shrimps during accli- mation which resulted in their protection. Conversely, if only the sea anemones containing acclimated shrimps failed to sting shrimps (either acclimated or un- acclimated), this would indicate that some change had occurred in the sea anem- one during acclimation, which resulted in the shrimp's protection (Mariscal, 1969, 1970a, 1971).

METHODS AND MATERIALS

Periclime/les rathbll/lae shrimps and S. helia/lthlls anemones were collected from Innes Bay, Port- land Parish, Jamaica, during December 1976, and transported to Earlham College, Richmond, Indiana where laboratory experiments were conducted in all-glass aquariums containing Instant Ocean brand synthetic sea water. Periclime/les a/lthophillis shrimps and C. giga/ltea anemones were collected during June and July 1978, from Whalebone Bay, Shelly Bay, Castle Harbor, John Smith's Bay, Harrington Sound, Baily's Bay, and North Rock, Bermuda. Laboratory observations were made on held in running sea water aquariums at the Bermuda Biological Station for Research. Sixty hours of field observations were made in Jamaica with the aid of snorkeling gear, and 16 h in Bermuda with the aid of SCUBA and snorkeling gear. Observations were made on the four experimental conditions mentioned above. Reciprocal exper- iments were conducted in which both acclimated and unacclimated shrimps were tested against their host sea anemone species with and without shrimp living with them (acclimated and unacclimated anemones, respectively). An acclimated shrimp was defined as a shrimp living with a sea anemone at the time of an experiment, and an unacclimated shrimp as one which had been isolated from anemones for 24 h. Shrimps were observed every 0.5 h for 5 minutes to determine if acclimation was complete. Criteria similar to those of Mariscal (1969, 1970a, 1971) were used to determine whether a shrimp was stung during experiments. The criteria were: strong tentacle adhesion to the legs, tail, and abdomen, a quick sudden jump backwards off the oral disc of the anemone when initially contacting the tentacles, and strong contraction of the anemone in response to the presence of the shrimp. The term "stung" is defined here as the action of spirocysts and nematocysts adhering to the exoskeleton that would result in the shrimp's capture if escape is not effected. Mariscal's (1969, 1970a, 1971) criteria for testing the anemones prior to each experiment were used to insure that they were capable of prey capture. These criteria included presenting live prey to the anemones, passing a finger through 462 BULLETIN OF MARINE SCIENCE. VOL. 30, NO.2, 1980

Table 2. Results of isolation experiments for Periclimenes anthophilus and its anemone host, Con- dy/actis gigantea

Anemone Anemone Without With Total No. of Shrimp Shrimp Shrimp Observations Summary of Results

Unacclimated Unacclimated + + (8) (20) 28 shrimps stung Acclimated Acclimated (10) (22) 32 shrimps not stung Total no. of observations 18 42 60

Numbers in parentheses refer to number of experiments + = shrimp stung; - = shrimp not stung.

the tentacles, which always resulted in strong adhesion, and passing gelatin-coated slides through the tentacles. The slides were examined microscopically for basitrichous isorhiza nematocysts. The shrimps were captured and transferred between aquaria using pyrex beakers. They were never taken out of sea water. Mariscal (1970a) found no difference in the results when fish or anemones were transferred. and therefore anemones were usually left in their aquaria and the shrimps were trans- ferred. Holthuis and Eibl-Eibesfeldt (1964) reported P. (/nthophilus in association with the anemone Actinia bermudensis McMurrich in Whalebone Bay, Bermuda. After preliminary field observations that did not concur with this report, a laboratory experiment was conducted to determine whether P. uwho- philus would associate with Actinia. Ten anemones and shrimps were placed in 11 cm diameter finger bowls, and observed every 0.5 h for 4 h.

RESULTS AND DISCUSSION Periclimenes rathbunae shrimps and S. helianthus anemones were found in the field at depths ranging from 0.3 to 3.0 m. One or two shrimps were found on a single anemone. Periclimenes anthophilus shrimps and C. gigantea anemones were found in the field at depths ranging from 1 to 15 m. The number of these shrimps on a single anemone varied from 2 to 11 individuals. Both species of shrimp were always found among the tentacles of their respective hosts, and the tentacles never reacted to the presence of the shrimps. The results of the isolation experiments with P. rathbunae are summarized in Table I, and those with P. anthophilus, in Table 2. Unacclimated shrimps were stung 100% of the time by anemones with and without shrimps living with them (P. rathbunae, n = 10 with, n == 10 without; P. anthophilus, n = 20 with, n = 8 without), indicating that a change had occurred on the part of the shrimps during acclimation. In all cases shrimps of both species swam directly to their respective hosts at the beginning of each experiment. Initial contact· of unacclimated shrimps with the oral disc of the anemone resulted in strong contraction on the part of the host, and strong tentacle adhesion to the legs, tail, and cephalothorax. In extreme cases, the shrimps made a quick backwards movement, broke away from the adhering tentacles, and jumped off the anemone. This occurred, for example, when the tentacles of C. gigantea encircled P. anthophilus and retracted, pulling the shrimp down towards the oral disc. During acclimation P. anthophilus con- stantly picked at the tentacles, apparently feeding. They often cleaned their other appendages with the chelipeds, and wiped the thorax and abdomen with the walking legs. Clear mucus was often seen adhering to their appendages, and presumably this was what they were "cleaning" off. Periclimenes anthophilus did not walk among t~l{::tentacles of C. gigantea continuously during acclimation, whereas P. rathbunae constantly moved around the oral disc of S. helianthus. As soon as P. rathbunae stopped walking, the LEVINE AND BLANCHARD: ACCLIMATION OF SHRIMPS TO SEA ANEMONES 463 anemone (S. helianthus) formed a depression around it and the shrimp had to tear loose from the adhering tentacles to avoid capture. Periclimenes anthophilus was able to remain stationary for periods of time without being encircled. Upon moving however, the anemone attempted to encircle it, and often the shrimp would have to break loose as described earlier. After acclimation, both shrimp species walked around the oral disc of their respective hosts unimpeded, and no tentacle adhesion was apparent. Periclimenes rathbunae acclimated to its host in 1 to 2 h, and P. anthophilus acclimated in 1 to 5 h. When the shrimps were fully acclimated to their anemone partners, the anemones no longer responded differentially to the shrimp's presence. This was verified in the field by repeatedly removing P. anthophilus shrimps from C. gigantea anemones and allowing the shrimps to return, and by stimulating the shrimps to move around among the tentacles (n = 20). All 10 of the P. anthophilus shrimps tested against Actinia anemones were stung initially upon contacting the tentacles. Eight of the shrimps avoided the anemones after being stung several times. One shrimp remained on the column of an anemone, and one other took up residence on the oral disc. In the latter case however, the anemone was not discharging nematocysts as determined by passing a finger through the tentacles. Clearly, these are not suitable hosts for P. anthophilus. The results suggest that P. rathbunae and P. anthophilus acclimate to their respective host anemones in a manner analogous to the acclimation of anemo- nefishes to their hosts (Davenport and Norris, 1958; Mariscal, 1966, 1969, 1970a, 1970b, 1971; Schlichter, 1967, 1968). It is possible that the shrimps become chem- ically camouflaged by acquiring an anemone mucus on their integument during acclimation. Schlichter (1972) found that anemones secrete more mucus when mechanically stimulated. It is plausible that an unacclimated shrimp would pro- vide sufficient stimulation to result in greater mucus secretion by the anemone. Autoradiographical studies similar to those of Schlichter (1972) need to be con- ducted with P. rathbunae and P. anthophilus to establish conclusively whether a substance is indeed transferred to the shrimp from the host anemones. Holthuis and Eibl-Eibesfeldt (1964) reported that when P., anthophilus walked on the tentacles of C. gigantea the anemone reacted to its presence by slowly contracting, especially when a shrimp was placed on an anemone which had not been inhabited for several days. While the tentacles were contracting they began to slowly encircle the shrimp, which responded by moving away. They further reported that when a tentacle touched a shrimp from above, the latter shook its antennae strongly and the tentacle moved away. They also observed that on one occasion a shrimp was "burned" and "had to free itself by force." The authors do not relate how they handled the shrimps; if a mucus coat is responsible for the protection of the shrimp, the manner in which they are handled is extremely important. Mariscal (1971) pointed this out in the case of anemonefishes. The experimental animals must be handled in a manner that does not alter or "damage" the mucus coating. Holthuis and Eibl-Eibesfeldt (1964) may have damaged the mucus coating on their animals, and if that was the case, their results are under- standable in light of the present study. The high variation in acclimation time, especially for P. anthophilus, may be due to two factors. First, 24 h may not have been sufficient time for all of the "mucus coating" to "wash" off. Varying amounts of mucus coating may have remained on the different test animals. A second factor concerns the condition (or composition) of the exoskeleton in terms of the intermolt cycle. Shrimps of both species that molted prior to the experiments appeared to be more vulnerable 464 BULLETIN OF MARINE SCIENCE. VOL. 30. NO.2. 1980

to the nematocysts. Their escape reactions were more pronounced in that they appeared to react to the anemone as vigorously when only lightly touching the tentacles with their legs as did shrimps coming into bodily contact (i.e., abdomen) with the tentacles. The third factor applies only to P. anthophilus. Condylactis gigantea, in comparison to other symbiotic anemones, is a weaker "stinger." When it is handled, one has the sensation of touching tacky paint. Stoichactis helianthus, however, is a powerful stinger. The tentacles often adhere to the hand, may tear from the anemone, and have to be scraped off. Since the shrimp symbiont of S. helianthus, P. rathbunae, is more likely to be severely stung at the beginning of acclimation than is P. anthophilus when acclimating to C. gi- gantea, P. rathbunae must move rapidly and continuously on the tentacles of the oral disc to avoid capture, whereas this is not the case with P. anthophilus. It is not understood why certain anemonefishes and shrimps cannot associate with any anemone. In the present study, both shrimp species showed a preference in the field for a specific anemone host. In both localities, anemone species that are known to be symbiotic partners of Periclimenes spp. in other localities, were not inhabited by shrimps. In Jamaica, the anemones C. gigantea and Bartholo- mea annulata (Lesueur) were present in great numbers, yet P. rathbunae were never found associated with them. It is interesting to note that P. rathbunae has been reported to associate with B. annulata in the Caribbean (Herrnkind et al., 1976; Mahnken, 1972). In Bermuda, the anemones L. danae and B. annulata were present in great numbers, yet P. anthophilus was never found in association with them. A comparison of other published studies on these shrimps is compli- cated by the fact that the taxonomic status of both is presently in doubt. In the case of P. rathbunae it is, as yet, unclear whether one polytypic species is in- volved, or whether two or even three species are hidden under the name P. rathbunae (F. A. Chace, personal communication). Periclimenes anthophilus has been distinguished from Periclimenes pedersoni Chace due to a slight variation in the carpus of the major second periopod, and because it associates with a different host anemone (Chace, 1972). The reason for the preference for hosts may lie in the inability of the guest symbiont (fish or shrimp) to acquire the mucus from other anemone species during acclimation. It is logical to assume that this mucus varies with the species of anemone. Further investigation is needed to determine the chemical composition of anemone mucus from different anemone species. There are probably similar- ities between the mucus of different species of anemones that could be acclimated to by a single species of a guest symbiont. Recent studies by Schlichter (1976) support these ideas.

ACKNOWLEDGMENTS

We wish to thank M. Dietrich, N. T. N. Reichardt, and H. F. Reichardt for assistance with translation of papers from the German; F. A. Chace, Jr., and R. B. Manning (Smithsonian Institution) for identification of shrimp species; the members of the Department of Biology at Earlham College for assistance, especially G. L. Ward; D. M. Ross for reviewing an earlier draft of the manuscript; M. L. Reaka and D. B. Bonar for their critical comments on the manuscript; J. J. McDermott for advice during the Bermuda part of the study and for reviewing an earlier draft of the manuscript; W. E. Sterrer for advice on localities in Bermuda; G. Bablanian, M. Hedrick, A. Leuenberger, and J. Manjarrez assisted in the collection of animals in Bermuda. The senior author is deeply indebted to R. E. Levine and C. S. Levine for their financial assistance and encouragement throughout the course of the study. This project was supported in part by the Joseph Moore Museum of Earlham College, and by a grant from the Exxon Corporation administered through the Bermuda Biological Station for Research. This is contribution No. 830 from the Bermuda Biological Station for Research, and contribution no. 942 from the Center for Environmental and Estuarine Studies, University of Maryland. LEVINE AND BLANCHARD: ACCLIMATION OF SHRIMPS TO SEA ANEMONES 465

LITERATURE CITED

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---, 1968, Das Zusammenleben von Riffanemonen und Anemonenfischen. Ztschr. Tierpsycho!. 25: 933-954. ---, 1970, Chemischer Nachweis, der Ubernahme Anemoneneigener Achutzstoffe durch Ane- monenfische. Naturwiss. 57: 3]2-313, ---. 1972, Chemische Tarnung. Die stoffliche Grundlage der Anpassung von Anemonenfischen an Riffanemonen, Mar. BioI. 12: 137-150, ---, ]976, Macromolecular mimicry: substances released by sea anemones and their role in the protection of anemone fishes. Pages 433-441 in G. O. Mackie, ed. Coelenterate Ecology and Behavior. Plenum Pub!. Corp., New York. Shoup, j, B, 1972, Notes on the behavior of Periclimenes imperator Bruce, an ectocommensal on the dorid nudibranch Hexabranchus marginatus Quoy and Gaimard (Decapoda, Palaemonidae), Crustaceana 23: 109-] I].

DATE ACCEPTED: March 13, 1979.

PRESENT ADDRESSES: (DML) Department of Zoology, University of Maryland, College Park, Mary- land 20742, University of Maryland, Horn Point Environmental Laboratories, Box 775, Cambridge, Maryland 21613 AND (OJB, Jr.) Department of Biological Sciences, Purdue University, West Lafay- ette, indiana 47907,