tt ADDITIONAL INFORMATION. IF ANY, CONCERNING AUTHORS, ADDRESS, TITLE, OR CITATION DATA PLEASE TYPE ABSTRACT DOUBLE SPACED BELOW — ALLEMAN, LANI L. (Hopkins Marine Station of Stanford University, Pacific Grove, California). Factors Affecting the Attraction of Acmaea asmi to Tegula funebralis (Mollusca: Gastropoda: Prosobranchia). The Veliger The behavioral basis of the association between Acmaea asm. and Tegula funebralis has been investigated. The observations indicate that A. asmi is not a cacted by a diffusible substance. but senses its substrate through contact with its tentacles. The critical substance(s) on the shell are easily destroyed or dis¬ solved by ethanol and slightly removed by distilled water, These factors are also present on Pagurus-inhabited Tegula shells, but are not found on uninhabited shells found on the beach, PLEASE DO NOT TYPE BELOW THIS LINt 18 C Factors Affecting the Attraction of Acmaea asmi to Tegula funebralis (Mollusca; Gastropoda; Prosobranchia) by Lani Lee Alleman Hopkins Marine Station Stanford University Pacific Grove, California,350 Toctusle1. Lani Alleman Acmaea asmi (Middendorff, 1849) lives almost exclusively as a commensal on the shell of Tequla funebralis (A. Adams, 1854). Pre- vious workers found this association to be quite specific, A. asmi preferring T. funebralis to Tegula brunnea (Philippi, 1848). Test (1945) suggested that T. funebralis released a chemical attractant. The source of this attractant was considered to be the shell by Radford (1959), whereas, Eikenberry and Wickizer (1960) concluded that both animal and shell were necessary. The following study attempts to settle these differences and to provide further infor- mation on behavioral and chemical aspects of this association. Materials and Methods Nost organisms used in this study were collected in inter- tidal areas near Hopkins Marine Station, except for Tegula brunnea Crmel Bag, Calig. which was collected in the Pebble Beach area, All experiments were run over night in pyrex dishes kept at 12-13°C in a darkroom. Preliminary experiments showed that if no choice were offered, Acmaea asmi would climb onto any shells tested. Therefore, to measure relative preferences, the limpets were allowed to choose between two different substrates. In the test five limpets were placed in about two om of water in the center of a 24 cm pyrex pie plate. Ten test shells were placed equidistantly around the periphery of the plate. There were five of each type to be compared, and they were placed alternately in the sircle. To permit washing of the Tequla shells with various solvents, the operculum was sealed with canning wax (Parowax). The animals survived this sealing treatment, and most importantly, were apparently O Lani Alleman unaffected by such solvents as alcohol or distilled water. In all experiments the dishes were then placed overnight in a darkroom at 12-13eC, and the number of limpets on the test shells determined the following morning. Results and Dissussion Rodford (1959) found that the shells of Tequla funebralis were not preferred by Acmaea asmi if the shells were boiled in alcohol for 15 minutes. The results shown in Figure lA indicate that simple room-temperature washing in ethanol for one hour also removes any attractant on the shell. In this experiment the limpets were given a choice between parowax-sealed shells washed in alcohol and control parowax-sealed shells. In five trials (25 limpets) the control shells attracted 90% of the limpets, while the alcohol-washed shells attracted ten per cent. The attractant is also partially removed by distilled water. If sealed shells containing Tequla funebralis are washed for two hours in distilled water and compared with normal, sealed shells, only 253 are found on the washed shells versus 65% on the control shells (rig. 18). (Where the total percentage does not equal 1007, the difference represents those animals not found on any shell.) However, distilled water washing is not as effective as alcohol washing. As seen in Fig. 1C, 60% of the animals preferred the water-washed shells as compared to only 15% on the alcohol washed. Although A. asmi is rarely found on Tequla funebralis shells inhabited by Paqurus spp., the limpets do not discriminate between these two types of shells. Thus, using the usual test system. Lani Alleman equal numbers of limpets were found on sealed T. funebralis shells containing Paqurus spp. as on Parowax-sealed T. funebralis shells containing its normal host (Fig. 10). Similar to normal shells, the attraction of Paqurus-inhabitated shells is lost when treated with alcohol (Fig. 1E). A final preference test series was made with old uninhabited Tequla shells found on the beach. Fig. Ir shows that Acmaea asmi prefers normal inhabited shells to these old shells. However, as shown in Fig. 16, treatment of normal shells with alcohol renders them as unattractive as the old shells. The above experiments suggest that the "attractant" is com- pletely removed or destroyed by alcohol, and partially removed or destroyed by distilled water. Furthermore, it is found on requla shells inhabited by either T. funebralis or Pagurug spp., but is not found on uninhabited shells found on the beach. The "attractant" then, could be an alga or bacterial film associated with the shell, ch is removed or destroyed by alcohol or distilled water. However, the major algal epiphyte found on T. funebralis is also found on the shells of Tequla brunnea and Acathina spirata (Eikenberry and Wickizer, 1964), which are not the normal hosts for Acmaea asmi Behavioral observations. The above results on preference are consistent with a diffus ible "attractant" emanating from the Tequla shell. Behavioral observations, however, indicate that the preference might be made at the tactile rather than olfactory or chemosensory level. Continuous observations were made of the selection process in Lani Alleman a test situation where normal, sealed Tequla funebralis shells were alternated with alcohol-treated ones. As soon as the Acmaea asmi were placed in the center of the Tequla funebralis circle, they extended their tentacles and began to feel the substrate. These tentacles are thin and almost as long as the shell when fully extended. They are moved in a tapping man- ner from side to side as the animal crawls in a seemingly random fashion across the bowl. When a specimen touched another A. asmi with its tentacles, it felt the shell and then climbed immediately onto it. If a limpet crawled between two Tequla funebralis shells, one alcohol-treated and the other not, it would tap each shell with its tentacles and then, in every case observed (11), climb onto the un¬ treated shell. If, however, the A. asmi encountered only one shell, whether alcohol-treated or not, it would generally climb on. Once on a shell, the A. asmi continued to move and sometimes changed shells. Out of twenty-five animals tested, nine were at one time or another on alcohol-treated shells. After eight hours, when the experiment was concluded, only two limpets were still on these shells. From these latter observations, it seems that Acmaea asmi is not reacting to some diffusible chemical attractant from the shell, but rather testing the substrates with its tentacles. It will crawl on the first curved surface encountered, but if other choices are available, will eventually end up on the preferred substrate. It is interesting to note that Test stated that the diffusible attrac- tant was sensed at seven mm and Radford felt the range to be ten mm. If an A. asmi is placed this close to a T. funebralig, it can generally Lani Alleman touch it with its tentacles and would, therefore, react to its presence SUN The behavioral basis of the association between Acmaea asmi and Tequla funebralis has been investigated. The observations indicate that A. asmi is not attracted by a diffusible substance, but senses its substrate through contact with its tentacles. The critical substance(s) on the shell are easily destroyed or dis- solved by ethanol and slightly removed by distilled water. These factors are also present on Paqurus-inhabited Tequla shells, but are not found on uninhabited shells found on the beach. Lani alleman ACKIOMLEDGENENTS This work was made possible by Grant G1806 from the Undergraduate Research Participation Program of the National Science Foundation. I also wish to thank Dr. David Epel, Dr. Donald P. Abbott, and Mr. Roger Kingston for offering helpful advice. 20 C Lani Alleman LITERATURE CITED Eikenberry, Arthur B., Jr., and Diane E. Wickizer 1964. Studies on the Commensal Limpet Acmaea asmi in Relatien to its Host, Tegula funebralig. The Voliger 6 (Suppl.): 66-70. Radford, Ruth 1959. A Study on Aomaes asmi. Unpubl. Stanford University honers paper. Test, Frederick 1945. Substrate and Mevements of the Marine Castroped Acmaes asmi. Amer. Midland Naturalist 33: 791-793 2 FOOTNOTES Footnote 1, page 1-Permanent address: Lani L. Alleman Lani Alleman Preference of A. asmi to various substrates. Twenty. Figure I. five A. asmi were tested in each experiment. Each test (A-H) represents the percentage of animals found on the indicated substrate (percentages of those not responding are not shown). C O onror (70%) . AlCONOA-TREATEO (10%) oNTot (65%) WATER-TREATEO (25%) Acotol-TREATEO (15% MATEE-TREATED (60%) Pas( T ses 18% TEGUA AN TEGOA SHE 30% Paeuels -eonreo (48%) Pbveus-Aonal (208 INHABITEL 76i sell(809) ONINAAÖTTED SES (0%) Reovor-reente 3) 6A WINANBTEDOD SHes () c