BULLETIN OF MARINE SCIENCE. 58(3): 668-674, 1996

REPRODUCTIVE BEHAVIOR OF THE CLIBANARIUS VI1TATUS (BOSC, 1802)

Brian A. Hazlett

ABSTRACT The precopulatory behavior patterns of the common intertidal hermit crab Clibanarius vittatus are described. Males precopulatory movements involve rotation of the female with the ambulatory legs and tapping lightly with one or both chelipeds in the aperture of the female's shell. Bouts of these courtship patterns alternate with period of guarding in which the female is held by the male's ambulatory legs with her shell aperture against the substrate. Females that copulated were not soft from a recent moult and brought out a new batch of eggs in less than an hour following copulation. Male-male competition for access to females was intense. The results of experiments demonstrated that courted females give off a pher- omone that stimulates male reproductive activity. Males occupying the sinistrally-spiralled shells of Busycon contratrium (Conrad, 1840) were at a distinct disadvantage in obtaining matings.

Descriptions of the reproductive behaviors of have often been used for comparative purposes (Ridley, 1983) and the precopulatory behavior of a number of of hermit crabs have been described (Hazlett, 1966, 1972, 1975). The reproductive behavior patterns of some members of the Clibanarius have been analysed both descriptively and to· some extent experimentally, e.g., C. tri- color and C. antillensis from the Caribbean (Hazlett, 1966) and C. zebra from Hawaii (Hazlett, 1989). However, none of the larger species of Clibanarius have been well described with regards to their precopulatory behavior. Existence of pheromones given off by one sex and detected by members of the other sex have been known for some decapods for a number of years (Ryan, 1966). However, clear evidence for a sex pheromone in hermit crabs has been lacking. Absence of a category of information in a major group of decapods, especially one that is reasonably well studied, might suggest something unusal about the courtship behavior of this group. In earlier studies of the mating behavior of hermit crabs it was suggested that the shape of the gastropod shell a male crab occupies could have strong effects on male mating success in some species. Crabs which are shell generalists, i.e., occupy a variety of shell species, had greatly reduced mating success in some shell types but this was not the case for crabs which were shell specialists (Hazlett, 1989; Hazlett and Baron, 1989). This suggestion of differences in resource use on reproductive behavior was based upon a limited number of crab species. The striped-legged hermit crab, Clibanarius vittatus is both common and geo- graphically wide-spread, occurring intertidally from Virginia, U.S.A., to Brazil (Young and Hazlett, 1978). It is an important member of the intertidal community in many areas and many aspects of its behavioral biology have been studied including: aggressive interactions (Hazlett, 1966, 1968a, 1974; Hazlett and Bos- sert, 1965), movements patterns (Fotheringham, 1975; Hazlett, 1981), responses to odors (Hazlett, 1968b, 1982; Katz and Rittschof, 1993), and shell utilization (Brooks and Mariscal, 1985; Fotheringham, 1976; Hazlett and Hermkind, 1980). Despite the number of papers on its behavior and behavioral ecology, the mating behavior of C. vittatus was observed on only one occasion (Hazlett, 1966) and has not been adequately described or characterized. The following observations and experiments were carried out in order to more

668 HAZLEIT: HERMIT CRAB BEHAVIOR 669 carefully characterize the mating behavior and reproductive activity of this com- mon, large, intertidal crab. In addition, because definitive proof of reproductive pheromones in hermit crabs has been lacking, the possibility of a female phero·· mane was examined in this species. The effect of the species of gastropod shell a male hermit crab occupied on its reproductive behavior was also examined.

METHODS

Mating Behavior.-Observations were made of precopulatory behavior of C. vittatus in both the field and the laboratory during June 1994 at the Duke University Marine Laboratory, Beaufort, N.C. While precopulatory behaviors were seen frequently in the field, the descriptions of reproductive activity are based primarily on the behavior of animals observed in the laboratory. Animals were maintained in water tables (67 cm by 123 cm, water 10 cm deep) supplied with running sea water and the bottoms of the water tables were covered with about three centimeters of sand. Precopulatory behavior was first noted on 6 June but had become infrequent, even with freshly collected animals, by 26 June. Very few instances of precopulatory behavior and no copulations were observed during July despite many hours of observations. In most cases, following observation of copulation, pairs were removed from the water table, isolated, and size and reproductive features recorded. The latter included the molt state of the female (whether her exoskeleton was soft or not), the presence or absence of a spermatophore, and the presence or absence of new eggs on the female's pleopods. Female Pheromone.-The rapid increase in activity of known males when some females were added to a water table suggested the possibility of a female pheromone. To test for a water-born substance given off by females, the following procedure was used. At the end of a day's observations, females that had been courted by males (see below) but had not been observed copulating were placed in a bucket in a different water table and supplied with running sea water and kept overnight (source of female water). Five large males werc kept overnight in another water table. The next morning, the water supply to the 2-gallon bucket containing the females (N = 4-5 depending upon mating activity) was shut off for 1 h as was the water supply to the water table containing the test males. Five smaller males were added to the male water table at the beginning of the hour to serve as possible objects to be grasped by the test males. A 2-gallon bucket containing five males was set aside (source of malt: water). During the control period of 5 min, water was siphoned from the male-containing bucket into one end of the water table at the rate of 200 ml per min. Each water table was divided into six sections by lines on the side and the locomotory rate of the test males was recorded as the number of lines crossed. The other behaviors recorded for the test males were the number of seconds spent by jndi,· viduals within two centimeters of the end of the siphon and the number of times the test males grasped the smaller males with their walking legs. The size differences between the test and smaller males greatly reduced the possibility that grasps were related to shell-exchange behavior. Following the introduction of "male water", the same behavioral parameters were recorded as water from the bucket containing females was introduced. Five replicates of this experiment were carried out and the results analyzed with paired t-tests comparing mean values of behaviors during the introduction of male and female water. Shell Species Occupation.-Observations suggested that the occupation of the sinistrally-spiralled shell Busycon contrarium adversely affected reproductive behavior in males (see below). The shell species occupied by large, reproductively active males in the water tables were recorded every several days during the weeks of observation. As a pilot study, five individual males which were observed to copulate when occupying other species of shell were removed from their original shells and placed in B. contrarium shells of the appropriate size. They were replaced in the water tables being observed and their behavior was followed for 2-3 days.

RESULTS Mating Behavior.-Mating activity was observed in the laboratory and in the field throughout June 1994. A number of matings (N = 10) and courtships (N =: 51) were seen during this month but during July only a very few courtship at·- tempts were observed. Thus this phase of reproductive activity seems to be quite constrained at least in the area around the Duke Marine Laboratory. Mating ac.. tivity was observed during both day and night although the majority of obser·- vations were made during the day. In most cases, the first indication of mating activity was the grasping of a 670 BULLETIN OF MARINE SCIENCE, VOL. 58, NO.3, 1996 female's shell by a male, The male initially got into an opposed position (Hazlett, 1966) with the female, the apertures of their shells facing one another. The male executed two types of behavior while holding the female in the opposed position: rotations and cheliped tapping. Rotations involved moving the female's shell from side-to-side in a figure eight fashion through an axis running dorso-ventrally through the aperture of the female's shell. The more common and persistent be- havior was cheliped tapping. The manus of the cheliped was held at a 90° angle to the carpus and the whole appendage moved up and down stiffly. Depending upon the relative size of the male to the female this involved either one or both chelipeds. If the male was not much bigger than the female both chelipeds of the male were raised and then quickly brought down simultaneously making light contact with the female's shell. The most common site "tapped" was the rim of the aperture of the female's shell, either just above or just below the aperture, partially in the aperture itself. If the male was somewhat larger than the female, such that both chelipeds could not easily fit in the aperture of the female shell, the male sometimes alternated single cheliped taps of the two chelipeds. In some cases, very large males persistently used just one cheliped or tapped primarily with just one or the other cheliped for a number of hours. In a few cases, as a cheliped was moved, the homolateral first ambulatory leg of the male was moved simultaneously, lightly tapping against the side of the female's shell. Bouts of rotation and cheliped tapping by the male alternated with periods during which the male manipulated the female's she]] such that the aperture of the she]] was against the substrate. In this "guarding" position, the male's am- bulatory legs closely encompassed the female's shell. Guarding was performed in bouts of 10-40 min. When there was a large size difference between the sexes, the female was sometimes difficult to see as the male completely covered the female's she]] with his body and ambulatory legs. In one case, a large male actually had two females under his body in the guarding position for a short period (before he lost one female to another male in a fight). Bouts of rotation and cheliped tapping alternated with guarding for a number of hours in most cases before any distinctive behavior by the female was observ- able. During rotation and tapping the female often was visible in the aperture of her shell and occasiona]]y the tapping appeared to very lightly touch the female's chelipeds or ambulatory legs. During both coursthip and guarding behaviors ag- gressive interactions between males were very common. Long male-male inter- actions involving multiple visual displays of the cheliped and ambulatory legs were sometimes fo]]owed by rapid movements of the appendages that appeared to hit the other male. It was not uncommon for these aggressive interactions to result in one male taking a female from another. After some hours of courtship behaviors by the male, the female moved further up in the aperture of her shell. The female palpated the mouthparts of the male with her chelipeds and ambulatory legs and this was followed by the male par- tially easing out of his shell into the copulatory position. In this position, the coxae of the male's fifth periopods were approximately opposite the coxae of the female's third periopods and the posterior portion of his cephalothorax depressed toward the female. This position was held for 10-30 seconds and the two animals then pulled back into their shells. The male usually then guarded the female for about 15 min following copulation. Females that were examined following copulation had massive spermatophores covering much of the ventral portion of the cephalothorax. The spermatophore mass was so large in some cases that all of the coxae of the female's walking legs were completely covered by it. When females were examined within 10-15 HAZLETT: HERMIT CRAB BEHAVIOR 671

Table I. Comparison of responses of males to introduetion of water from males or from females that had been courted. Means (SE) of the five replicate tests.

Male water Female water (-test P value No. of Grasps of small shells by males 1.2 (0.8) 6.8 (1.0) 6.3 0.008 No. Lines eros sed 7.0(3.1) 14.5 (3.1) 4.6 0.010 No. Seeonds near siphon 6.0 (3.9) 304.0 (96.8) 3.1 0.035

min post-copulation they usually did not have any eggs on their pleopods and none of the courted females were soft from a recent moult. Females examined 30-60 min after copulation had either a partial or full complement of new eggs on their pleopods. The most striking exceptions to the pattern described above were two of the females that had been prevented from copulating overnight as part of an experi- ment on female pheromones (see below) and then reintroduced to males. Upon placement with males, each of these females went very directly to the nearest male and initiated palpation of his mouthparts. The male in each case grasped the female, gave one or two cheliped taps and then copulated. Both of these females had eggs extruded when examined just 15 min after copulation although one of them copulated with two males before she was examined. Of the 10 copulations observed, the male was larger than the female in nine cases (25% to 107% greater in cephalothorax length). In the one case of the female being larger, she was only 9% larger in cephalothorax length. In a number of instances among the 51 courting pairs observed, the males were not markedly larger than the female but in almost every case these relatively smaller males were displaced aggressively by larger males. Long aggressive interactions involv- ing both visual displays as well as pushing and striking with both chelipeds and walking legs were commonly observed as males courted and guarded. In more than one instance males were engaged in aggressive activity and the female (being fought over) simply walked away from the fighting pair when she was not care- fully guarded. Female Pheromone Results.-When female water was introduced to a tank of males, there was a significant increase in locomotion (Table I) compared to male water. In addition, there was a significant difference in the number of times that small males in the tank were grasped by large males when female water was added. The time spent by males in the immediate vicinity of the siphon intro- ducing the water was significantly greater for female water than for male water. Male Shell Occupation Effects.-A total of 51 pairs of crabs were observed court- ing in the laboratory and copulation was observed in 10 of these pairings. As shown in Table 2, the proportion of courtships and copulations by males in Fas-

Table 2. Number of courships and eopulations observed categorized by shell species occupied by males. (The proportion of shell species occupied in the water table was determined by periodic counts of the males in the water tables under observation)

Proportion in Shell species occupied water tables Courtships observed Copulations observed

Busycon contrarium 48% 6 (12%) I (10%) Busycon carica 29% 34 (66%) 7 (70%) Fascio/aria tu/ipa 14% 5 (10%) 1 (10%) Po/inices dup/icatus 10% 6 (12%) 1 (10%) 672 BULLETIN OF MARINE SCIENCE. VOL. 58. NO.3. 1996 ciolaria tulipa (Linne, 1758) and Polinices duplicatus (Say, 1822) were similar to the proportions expected by their frequency in the laboratory population of large males. In contrast, males in Busycon contrarium were less frequently seen courting or copulating than expected and males in B. carica (Gmelin, 1791) were more frequently seen courting and copulating. The distributions of both courtships (chi-square = 40.9, P < 0.01) and copulations (chi-square = 6.5, P < 0.025) were significantly different than those expected from the frequency of occupation of the four species of shells. The proportions of males in the four shell species that were observed copulating were clearly very similar to the proportions ob- served courting. The only male in a B. contarium shell that successfully copulated with a female was the largest male in the water tables (el = 40.4 mm) and had successfully copulated the day before when in a B. carica shell, before I moved him into a B. contrarium shell. The other four males that had been moved into B. contrarium shells following successful copulation failed to gain a second copulation on other days of observation. The failure of males in B. contrarium shells to court appeared to be partially a result of them spending more time engaging in shell exchange behaviors. Moreover, in several cases where males in that species of shell did engage in courtship activity they held the female in unusual positions during courtship, 60 to 90° out of the opposed position while trying to execute courtship behaviors. While not quantified, it appeared also that males in B. contrarium shells tended to lose females to males in other shell species during aggressive interac- tions more frequently than would be expected for the sizes of the males.

DISCUSSION The precopulatory behavior patterns shown by individuals of Clibanarius vit- tatus resembled those of other diogenid hermit crabs (Hazlett, 1966). In particular, in other Clibanarius and species, males often alternate rotations of the female with tapping movements of the chelipeds. The dominance of tapping by males of C. vittatus and the stiffly held nature of the appendage as it is moved up and down are distinctive. Even more distinctive is the tending or guarding behavior of males which alternated with the courtship patterns. Guarding in other decapods has been described (Hartnoll, 1969), but a distinctive guarding posture has not been observed in diogenids. While periods of active precopulatory move- ments may alternate with periods of the male quietly holding the female in the opposed position (Hazlett, 1966), the pattern of the male turning the female over and surrounding her shell with his ambulatory legs has been observed only in this species. This may be correlated with the rather narrow courtship season in C. vittatus compared to some other species where courtship has been observed over longer periods of time (Ameyaw-Akumfi, 1975; Hazlett, 1966; Reese, 1968) although often in more tropical waters. Another aspect of the reproductive be- havior that would favor guarding is the fact that females are very ready to bring out new eggs when they are being courted, i.e., fertilization is probable in the very near future. The fact that none of the courted or copulated females was soft from a recent moult is not unique among diogenid hermits but does contrast with the pattern seen in some other species. Females of C. tricoLor were always soft from a recent moult when courted (Hazlett, 1966) while in hummi, females could be either soft from a recent moult or not (Hazlett, 1972). The speed with which females brought out new eggs following copulation in C. vittatus was impressive. In CaLcinus tibicen, new eggs were not in place until 5-10 hs after copulation HAZLETT: HERMIT CRAB BEHAVIOR 673

(Hazlett, 1966). It appears that females are strongly physiologically primed when they respond to male courtship by signaling the male and easing out of their shell for copulation. The existence of female sex pheromones has been demonstrated in a number of decapod such as portunid crabs (Gleeson, 1980), homarid lobsters (Atema, 1986), and freshwater crayfish (Ameyaw-Akumfi and Hazlett, 1975). The responses of male Pagurus bernhardus (Linne, 1758) to a cloth bag containing a recently courted female were suggestive of a chemical cue being given off by females (Hazlett, 1970), however, a similar experiment with individuals of C. tricolor yielded no evidence of such a chemical (Hazlett, 1966). The results of the "female water" experiments reported here were so clear that a limited number of replicates was adequate. Male C. vittatus clearly showed an increase in both general activity and specific courtship behaviors when exposed to water contain- ing recently-courted females. Further experiments are needed to see if males were receptive to female water only after females have been courted for a set length of time. Individuals of C. vittatus will occupy sinistrally spiraled shells such as Busycon contrarium. Given the morphological asymmetry of the abdomen present in C. vittatus and most other hermit crabs, which facilitates use of dextrally spiraled shells, it is not surprising that males would have some difficulties behaving ef·· fectively in such a shell. While the "problems" that males had while trying to court females and defend them appeared subtle, they seemed to have very strong effects on the chances of a male obtaining matings. This is very similar to the situation with other shell generalists, such as Calcinus tibicen from the Caribbean and Clibanarius zebra Dana, 1852 from Hawaii, where occupation of certain shaped (round apertured) shells resulted in very strong effects on the chances of mating in those species (Hazlett, 1989; Hazlett and Baron, 1989) reducing mating chances almost to zero in some cases. In those species, the failure of males to successfully court females when in certain species of shell seemed mostly due to females not responding to male behaviors, while in C. vittatus the primary prob- lem appeared to be the inability of males in B. contrarium shells to defend females against other males.

ACKNOWLEDGMENTS

This work was made possible by support from the Cocos Foundation and Duke University. Special thanks are given to the staff of the Duke Marine Laboratory and to D. Rittschof for his help with the research. The manuscript was improved by the comments of R. Thacker and C. Bach.

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DATEACCEPTED:February 3, 1995.

ADDRESS:Department of Biology, University of Michigan, Ann Arbor, Michigan 48]03.