Deborah S. Yokoe INTRODUCTION Crepidula adunca (Sowerby, 1825), commonly known as the slipper shell, is a protandric marine prosobranch commonly found intertidally on the shells of the black turban snail Tegula funebralis (A. Adams, 1854). Its range extends from Vancouver, British Columbia, to the tip of Lower California. Crepidula adunca females brood a mass of yolky eggs until developed and will physically push the young into the surrounding water (Putnam, 1964). The young lack sexual organs and after a growth period develop a penis and enter the male phase of their life cycle. At some point, dependent partly upon environmental factors, the Crepidula enter the female stage of their life cycle (Coe, 1936). The male is typically found oriented on the female shell which in turn is oriented on the T. funebralis shell (Coe, 1953). Crepidula adunca reportedly use a radula for rasping algal films from rocks when young and switch to a filter- feeding system as they mature into adults (Coe, 1948; Abbott and Haderlie, 1980). The results of this investi¬ gation suggest that in terms of feeding behavior, both young and adults may be more labile than previously believed. Newborn Crepidula adunca have a mean length of about 1.18 mm and are very similar to the adults in appearance. Essentially nothing was known about the period of the Crepidula life cycle between the time of dispersal and the time of attachment to Tegula. Although newborn Crepidula are about 1 mm in length, previous investigators (Putnam, Deborah S. Yokoe 1964) were unable to find Crepidula adunca smaller than 3.8 mm in length in the field. This investigation was focused towards solving the mystery of the habitat of the young after dispersal. The results obtained suggest that young Crepidula adunca attach to small gastropod shells in sandy, shell-fragmented areas neighboring the rocky areas where the adults are found on Tegula funebralis. MATERIALS AND METHODS I. Comparison of young and adults A Crepidula hatchery was set up by placing Tegula with male-female Crepidula pairs in aquaria and collecting the young as they were dispersed. The first phase of this investigation involved comparison of the behavior and desiccation tolerance of young and adult Crepidula through experimentation in the laboratory. The motility of young was compared to that of adult males and females by measuring the distances from the point of origin of individual Crepidula over intervals of five minutes in a large glass dish. Vulnerability to desiccation was tested by removing the animals from water, placing them on a glass surface, and examining them periodically for response to probing. Death was defined operationally as failure to respond to probing stimuli after reimmersion in seawater for 30 minutes. The feeding behavior of the young was compared to that of the adult by observing radular rasping action or filter-feeding. To test for radular feeding, a Crepidula was placed on a diatom-covered slide turned upside-down into a shallow dish of water and observed under a dissecting microscope for scraping of diatoms by the radula. In order Deborah S. Yokoe to determine whether an animal was filter-feeding, it was placed on a clean microscope slide, placed upside-down in a shallow dish of water with a few drops of graphite solution, and examined under the dissecting microscope for the appearance of a graphite-blackened mucus string travelling to the mouth up the ciliated food groove located on the right neck lappet. An artificial microhabitat was set up in the laboratory by placing a layer of sand covered by shell fragments and rocks in a large glass dish along with gastropods and Pagurus in gastropod shells ranging from 2 mm to 20 mm in length. 20 newborn Crepidula were added, and the micro- habitat was left undisturbed for a week. II. Field Studies Areas of high adult Crepidula density and the surrounding regions of the intertidal were examined for the presence of young Crepidula. Distribution of Crepidula on hosts of adult Tegula funebralis on rock faces was surveyed by collecting each sample within a haphazardly placed quadrant of area of 1 m2. Distribution of Crepidula on host gas- tropod shells of sandy bottoms was surveyed by collecting samples of approximately equal volumes from the bottom in a metal scooper. III. Attraction of Crepidula to T. funebralis Preference tests were performed between live T. fune¬ bralis and empty T. funebralis shells, between T. funebralis funebralis shells, and between T. fune- and Pagurus in T. bralis shells and T. funebralis which have had their outer black layer sanded off. These tests involved placing Deborah S. Yokoe a male Crepidula in the center of a glass dish with, for example, a live Tegula and a Tegula shell, at opposite sides of the dish and examining the attachment of the male Crepidula after 24 hours. Experiments to examine the possible protection from predation by attachment of Crepidula to a Tegula shell were performed by placing 5 male Crepidula, 5 T. funebralis, and 5 Pagurus in T. funebralis shells together in a glass dish and observing survival rate of Crepidula over time. ESULT The motility experiments (Fig. 1) show that the young, which although in body length are an average of 10 times smaller than the females and 7 times smaller than the males, ex- hibit greater movement over time than either the males or females. The young show an average distance moved over 30 minutes of 3.18 cm, the adult males show an average distance moved of 2.16 cm, and the adult females show an average distance moved of 2.04 cm. Desiccation experiments showed the young to be much more vulnerable to desiccation than the adults. When placed out of water, the young die within 15 to 20 minutes while the adults are able to survive for several hours. Observation of feeding behavior of the newborn indicate that Crepidula adunca are born not only with the ability to rasp but also with the capacity to filter-feed. Although the food pouch does not develop until after the young adult stage (Moritz, 1939), a mucus string travelling up the Debor ah S. Yok food groove to the mouth was clearly observed in the newborn. Similarly, although adults reportedly lose their ability to feed by rasping, a definite scraping by the radula of diatoms off of the slides was observed not only by the young but also by both adult males and females. After an interval of a week, the microhabitat which had been constructed to resemble the sandy, shell-fragmented habitat of the young, resulted in a clustering of newborn Crepidula on the smallest gastropod shells available. 19 out of 20 were found on shells smaller than 8 mm in length and no Crepidula were found attached to adult T funebralis. Examination of rock faces and crevices where adult Crepidula are abundant yielded no Crepidula smaller than 4 mm in length. Examination of nearby sandy, shell-frag- mented areas and sandy holdfasts of algae and Phyllospadix revealed a number of small gastropod shells and Pagurus in small gastropod shells with Crepidula attached which ranged down to the 1 mm size of newborn Crepidula (Fig. 2). The results of the distribution survey in the lower, sandy, shell-fragmented habitats are shown in Fig. 3, in which the number of host shells, host shells with Crepidula, and host shells with Crepidula of 1 mm in length for each host shell size class are shown. The abundance of small gastropod shells and the clustering of the newly born Crepidula on the smallest gastropod shells are apparent. The average size of Crepidula found is 2.00 mm with a standard error of .18. The average size of host shells with 1 mm Crepidula attached is 3.90 mm with a standard error of .32. The average Deborah S. Yokoe size of shells with Crepidula attached is 6.67 mm with a standard error of .61. The average size of all potential host shells is 7.02 mm with a standard error of .27. 18.1% of all potential host shells had Crepidula attached. In contrast, the distribution of shells in the rock face habitat shows an abundance of large host shells and a com¬ plete absence of Crepidula of 1 mm (Fig. 4). Average size of Crepidula found is 8.25 mm with a standard error of .52. The average size of all potential host shells is 15.74 mm with a standard error of .17. The average size of host shells with Crepidula attached is 17.18 mm with a standard error of .32. 13.2% of all potential host shells had Crepidula attached. The data indicate that there is a clustering of small Crepidula and gastropod shells and a correspondence between size of Crepidula and size of host shell in the sandy habitat which are lacking in the rock face habitat. Fig. 5 shows the relationship between size of Crepidula and size of host shell found in the sandy habitats. A least squares linear regression was performed and yielded the line y-.19 +.28x which appears on the graph. A Pearson R correlation coefficient of R=.81 was obtained, indicating a strong correlation between Crepidula and host shell sizes. Also apparent is an increasing variability in Crepidula sizes found on host shells with an increase in host shell size. In contrast, size of Crepidula versus size of host shell in the rocky habitats (Fig. 6) show essentially no correlation, a large range of Crepidula sizes found on all host shell sizes present. Also of note Deborah S. Yokoe is the absence of small host shells and Crepidula smaller than 4 mm. The results of the preference tests performed with adult Crepidula over an interval of 24 hours indicate that adult Crepidula showed no preference between T. funebralis and empty T. funebralis shells (P».05), no preference between T. funebralis and Pagurus in T. fune- bralis shells (P).05), and no preference between T. fune¬ alis shells and T. funebralis shells which have had their outer layers sanded off (P).05). Predation experiments resulted in a 100% survival rate of Crepidula. Within about 15 minutes all Crepidula had attached to either a T. funebralis or Pagurus in T. fune- bralis shell. Even after 2 weeks of starvation, the Pagurus made no attempt to remove Crepidula from Tegula shells. DISCUSSTON Field studies indicate that young Crepidula adunca are found in sandy, shell-fragmented areas and among the sandy holdfasts of algae and Phyllospadix neighboring the rocks of the adults.They are on small gastropod shells including juvenile T. funebralis and small gastropod shells occupied by Pagurus. This habitat seems to fit in well with the information known about newborn Crepidula. By attaching to organisms which prefer sandy, lower habitats almost constantly immersed in water, the young Crepidula are able to avoid conditions conducive to desiccation which the adult Crepidula on T. funebralis, often found (Wara and Wright, 1963), are able to with- out of water Deborah S. Yokoe stand. The shell fragments and blades of algae characteris¬ tic of the habitat of the young may provide protection from strong water currents which might tend to tear the young Crepidula away from the host shell. Putnam (1964) found that newly dispersed young, because of their inability to free swim, will fall off the host shell and sink. This characteristic of the young would provide an effortless means of transportation between the rock face habitat of the adults and the sandy bottom habitat where the young are found. The ability to both rasp and filter-feed indi- cated in laboratory observations would grant the young more flexibility in habitat choice. The strong positive correlation between size of Crepi- dula and size of host shell in the sandy habitat seems to suggest that very small Crepidula will attach to a small gastropod shell until it outgrows it, at which point it will move on to a bigger host shell. At some point in its life cycle, however, Crepidula seem to develop a strong attraction for the Tegula funebralis and female Crepidula shells and will settle down on one host for long periods of time. This prolonged attachment of male and female Crepidula of various ages would result in the wide range of Crepidula sizes found on the adult T. funebralis shells of the rocky habitats. The relatively high motility of the young fits in well with the possibly higher rate of host exchange of the young indicated in the laboratory. The lack of attraction towards adult T. funebralis or Deborah S. Yokoe 10 Crepidula adunca in the young and strong attraction of the adult observed by Putnam (1964) would also fit in well with the transition from sandy habitats where Crepidula are found attached to a variety of small shells to rocky habitats where T. funebralis are found almost exclusively. The attachment behavior of the young in the artificial microhabitat also confirms the preference of newborn Crepidula for small gastropod shells. The predation experiments suggest that attachment of Crepidula to Tegula shells may serve as a means of protec- tion from Pagurus predation. The preference experiments were designed to explore the question of what actually attracts the adult Crepidula adunca to the host T. funebralis shell. The lack of preference between live Tegula and empty Tegula shells and between Tegula and Pagurus in Tegula shells both seem to imply that the main attracting agent is not the live T. funebralis animal. The lack of preference between scraped and unscraped T. funebralis shells indicates that the outer material and texture of the shell does not serve as the main attrac¬ tant. The shape of the Tegula shell may be the factor which triggers the attachment behavior of the Crepidula. Rolfes and Turken (1979) found that Crepidula adunca exhibited a definite host species preference for T. brunnea and T. funebralis over T. montereyi which corresponds to a gradient in shell roundedness. This finding suggests that Crepidula may use host shell shape as a criterion for attachment. If this is the case, an increased sensitivity to host shell Deborah S. Yokoe 11 shape may be the cause of attraction of Crepidula for T. funebralis and together with the selection of larger host shells may result in the change of habitat to the rock faces where Tegula are abundant. Thus, the results of motility, desiccation, and micro- habitat experiments as well as observations of feeding and dispersal behavior harmonize well with the discovery of the sandy, shell fragment habitats of young Crepidula adunca below the neighboring rocky habitats of the adults and with the observation that the young prefer small gastropod shells as their hosts. A marked difference in the behavior of the young in subtidal populations may exist. Young Crepidula adunca are reported to have been found on adult Tegula species common to the subtidal region. This may possibly be due to the difference in habitat conditions, such as the lessened threat of desiccation in the subtidal areas, or possibly even due to genetic differences between intertidal and subtidal populations of Crepidula adunca. Although the question of where intertidal Crepidula adunca go after dispersal has been partially answered by this investigation, a whole range of questions yet to be answered have arisen. SUMMARY The young of intertidal Crepidula adunca are dispersed from the female and sink down into sandy, shell-fragmented, lower habitats where they attach onto small gastropod shells. As they outgrow the host shell, they move onto Deborah S. Yokoe larger gastropod shells, producing a strong correlation between size of Crepidula and size of host shell. At some point in their life cycle, the Crepidula experience a strong attraction towards Tegula funebralis shells which does not involve the actual animal or the shell surface material or texture but rather possibly an increased sensitivity and/or attraction towards Tegula shell shape. At this point the Crepidula attach to adult T. funebralis which prefer rock faces and crevices. It is in these rocky habitats that the adult Crepidula are found most abundantly in the inter- tidal region. Deborah S. Yokoe TEE ACKNOWLEDGEMENT The results of this investigation are due in large part to the guidance and support of Charles Baxter. Thanks is also due to Dr. Donald P. Abbott for background information concerning Crepidula adunca, to James Watanabe for use of equipment and subtidal expertise, and to Mike Rothman, Richard Peters, and Peter Gerbino for invaluable assistance during the quarter. Deborah S. Yokoe LITERATURE CITED Abbott, Donald P. and Haderlie, Eugene C. 1980. Prosobranchia: Marine Snails, Chapter 13 in Morris, Abbott, and Haderlie Intertidal Invertebrates of California. Stanford University Press, Stanford CA, 928 pp. Coe, Wesley R. 1936. Sexual phases in Crepidula. J. Exp. Zöól., Philadelphia. 72 (3): 455-476. Coe, Wesley R. 1948. Nutrition and sexuality in protandric gastropods of the genus Crepidula. Biological Bulletin. 94 (2): 158-160. Moritz, C.E. 1938. The anatomy of the gastropod Crepidula adunca Sowerby. University of California Publications in Zöölogy. 43 (5): 83-92. Putnam, Deborah A. 1964. The dispersal of young of the commensal gastropod Crepidula adunca from its host Tegula funebralis. The Veliger 6 (Supplement): 63-66. Rolfes, Richard H. and Turken, David S. 1979. Factors affecting the distribution of Crepidula adunca on the host genus Tegula. Unpublished Hopkins Marine Station paper. Deborah Yokoe 15 Wara, William M. and Wright, Benjamin B. 1964. The distri- bution and movement of Tegula funebralis in the intertidal egion of Monterey Bay, California. The Veliger 6 (Supplement): 30-37. FIGURE CAPTIONS Figure 1: Comparison of the distance covered by young, adult males, and adult females over a 30 minute period. Figure 2: Comparison of the habitat locations of young and adult Crepidula adunca. Figure 3: Number of shells, number of shells with Crepidula attached, and number of shells with Crepidula of 1 mm attached for each host shell size class in the sandy habitats of the young. Figure 4: Number of shells and number of shells with Crepidula attached for each host shell size class in the rocky habitats of the adults. Figure 5: Relationship between size of Crepidula and size of host shell found in sandy habitats. Least squares linear regression line is shown. Figure 6: Relationship between size of Crepidula and size of host shell found in rocky habitats. Figure 7: Results of preference tests performed with male Crepidula. Young 0 — 10 Adult Males 3 6 2 a1 — taaa- 10 Adult Females 2 15 20 25 30 10 TIME (min) 2 ADULT HABITAT rock face water holdfast shell fragments sand HABITAT OF YOUNG 80 72 64 56 48 5 32 2 24 16 shells, Sshells with Crepidula shells with Crep. of Imm 2 4 6 8 10 12 14 16 18 20 HOST SHELL SIZE (mm) 80 72 64 56 48 32 224 16 shells shells with Crepidula .. . . 2 4 6 8 10 12 14 16 18 20 22 HOST SHELL SIZE (mm) 20 18 14 412 o u 8 6 3 . . 223. zer:. 2 4 6 8 10 12 14 16 18 20 SIZE OF HOST SHELL (mm) 20 19 18 17 14 13 E 12 o. 8 . ** * . 3 3 . 3 3 . 3 . . 3 3 3 . . e . . 3 * 3 . 3 . 2 4 6 8 10 12 14 16 18 20 22 SIZE OF HOST SHELL (mm)