Effects of Visual and Chemical Cues on Orientation Behavior of the Red Sea

The Journal of Basic & Applied Zoology (2012) 65, 95–105

The Egyptian German Society for Zoology The Journal of Basic & Applied Zoology

www.egsz.org www.sciencedirect.com

Eﬀects of visual and chemical cues on orientation behavior of the Red Sea hermit crab Clibanarius signatus

Tarek Gad El-Kareem Ismail

Zoology Department, Faculty of Science, Sohag University, P.O. Box 82524, Sohag, Egypt

Received 17 December 2011; accepted 4 February 2012 Available online 24 September 2012

KEYWORDS Abstract Directional orientation of Clibanarius signatus toward different targets of gastropod Visual cues; shells was studied in a circular arena upon exposure to background seawater, calcium concentra- Chemical cues; tions and predatory odor. Directional orientation was absent when crabs were presented with the Orientation; white background alone. Each shell was tested in different positions (e.g., anterior, posterior, Hermit crab; upside-down, lateral). Adult crabs were tested without their gastropod shells, and orientation varied Red Sea with concentration and chemical cue. With calcium, orientation increased as concentration increased up to a maximum attraction percentage and then attraction became stable. In the case of predator cues, some individuals swim away from the target toward the opposite direction representing a predator avoidance response. Whenever, the blind hermit crab C. signatus was exposed to a shell target combined with calcium or predator cues, the majority of them stop moving or move in circles around the arena center. The others exhibited uniform orientation distribution. The responsiveness was higher with calcium cues than predator cues. Thus in the absence of vision, individual hermit crabs were able to detect both calcium and predator cues and have different response regarding them. ª 2012 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved.

Introduction between predators, conspeciﬁcs and refuge (Woodbury, 1986; Romano et al., 1990; Chiussi, 2002) while others use chemical Directional orientation is regarded as one of the major behav- cues for orientation since aquatic organisms live in a complex ioral mechanisms required for survival of motile crustaceans cocktail of chemical stimuli (Bro¨ nmark and Hansson, 2000). living in intertidal areas (Diaz et al., 1995a). Many crustaceans Thus, chemical stimuli are a major source of information for use visual cues for orientation and are able to discriminate be- many invertebrates about their aquatic environment especially tween shapes providing them with the ability to differentiate those living in topographically complex habitats such as intertidal areas (Iglesias, 2007; Turra and Denadai, 2002; Dill, 1987; Chivers and Smith, 1998; Kats and Dill, 1998). Different E-mail address: [email protected] stimuli elicit different behaviors including aggregation, spawn- Peer review under responsibility of The Egyptian German Society for ing, attraction to mates, searching for food, homing in suitable Zoology. microhabitats and appropriate responses to potential predators, such as hiding or ﬂeeing. Therefore, at any particular mo- ment, an individual must not only be a monitor of these Production and hosting by Elsevier diverse stimuli, but also rank the value of potential behavioral

2090-9896 ª 2012 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jobaz.2012.02.004 96 Tarek Gad El-Kareem Ismail responses to decide which response is most appropriate (Dill, mangroves are mixed with the impressive remains of fossilized 1987; Kats and Dill, 1998; Dicke and Grostal, 2001). corals. Hermit crabs make an ideal model system for studying sensory capabilities and decision-making processes in crustaceans, Hermit crab collection and maintenance because their shelters, food sources and mates may all poten- tially have the same appearance. This may necessitate the The hermit crab, C. signatus (Heller, 1861), is a common spe- adaptation of behavioral and physiological means to differen- cies within the mangrove site and has a spatial distribution in tiate between resources. To make efficient use of information, the intertidal zone (El-Wakeil et al., 2009; Ismail, 2010, 2011). it must be sorted or prioritized (Billock and Dunbar, 2009). In July 2009, large numbers of hermit crabs were randomly Hermit crabs tend to use visual and chemical cues for orien- hand-collected around mangrove roots at daylight and low tation (Diaz et al., 1995a; Chiussi et al., 2001), agnostic inter- tide. The selected crabs were occupied in different shell types actions (Briffa and Williams, 2006), predator avoidance (see Ismail, 2010). The hermit crabs were separated into small (Brooks, 1991; Rotjan et al., 2004; Chiussi et al., 2001), con- groups and transferred to the laboratory. In the laboratory, specific identification and shell location (Rittschof et al., they were maintained in aerated seawater arenas (20 L) in 1992; Gherardi and Tiedemann, 2004; Gherardi et al., 2005). groups of about 40 individuals under a natural 14:10 light:dark Moreover, hermit crabs are known to modulate their behavior cycle and a water temperature of about 31 ± 2 C. Hermit in response to surrounding visual and chemical cues (Pezzuti crabs were extracted from their original shell by gently break- et al., 2002; Mima et al., 2003; Gherardi and Atema, 2005; ing it with a bench vise, sexed and measured to the nearest Briffa and Williams, 2006). 0.1 mm. Only sexually mature males with cephalothorax Most hermit crab species need shells for nearly all aspects of lengths ranging between 11.3 and 12.5 mm were used in the their biology (Laidre and Elwood, 2007) and use shell seeking present investigation to avoid any bias in results due to size, behavior to locate gastropod shells which become available maturity variations and sex (to avoid the possibility of chemi- due to death of a gastropod or conspecific (Rittschof, cal cues associated with reproduction) (Rodrigues et al., 2002; 1980a,b). Hermit crabs can locate shells visually (Ohta, 1971; Briffa and Williams, 2006). The hermit crabs were fed with a Kinosita and Okajima, 1968; Orihuela et al., 1992) and dis- diet of commercial fish pellet every day, and water was chan- criminate their shapes (Diaz et al., 1994, 1995a) which play ged every 2 days. an important role in shell identification and selection (Hazlett, 1975; Salmon and Hyatt, 1983; Chiussi, 2002). Also, hermit Experimental design crabs use chemical cues for guidance to sites of shells that are associated with predation events (Gilchrist, 1984; Rittsc- Five series of experiments were performed in the present inves- hof, 1980a,b), and death of a conspecific (Rittschof et al., tigation; (1) orientation of the crabs toward a solid target in 1992). It is believed that many species switch from chemical ambient clean sea water, (2) orientation of the crab individuals cues at long distances to visual cues at short distances (Bell, toward different views of varying shell species subtended at a 1991). Although either visual or chemical cues may be used fixed angle in ambient sea water, (3) orientation of the crab independently for orientation toward shells (Chiussi et al., individuals toward a fixed-angle target in the presence of cal- 2001), integration of input from both senses may determine cium cues, (4) orientation of the crab individuals toward a responses(Hazlett, 1982). Underwater, hermit crabs respond fixed-angle target in the presence of predator cues and (5) ori- to silhouettes of shells or predators either by moving toward entation of the blinded-eyes crabs toward a fixed-angle target or away according to their odors and visual images (Mesce, in the presence of different previous two chemical cues. 1982; Hazlett, 1982; Diaz et al., 1995a). All experiments were done in translucent circular plastic The present work aims to; (1) examine visual orientation of arenas (54 cm diameter) surrounded by an opaque white wall Clibanarius signatus when confronted with targets subtended of 16 cm height. The arenas were held within a small indoor at different angles, (2) test if C. signatus can visually discrimi- room to ensure similar activity levels of test species (Iglesias, nate between shells from different species of gastropods, (3) 2007). The arenas were filled to a depth of 7 cm with ambient determine if orientation response of C. signatus changed in sea water (salinity about 39.5%) or ambient sea water condi- the presence of chemical cues of calcium and a predator and tioned by two chemical cues. All experiments were conducted (4) and to determine whether olfaction or visual has greater between 08:00am and 18:00pm. Illumination during observa- influence on hermit crab response and orientation. tions was provided by a 75-W incandescent light, 50 cm above the water level. After each experiment, crabs were caged indi- Materials and methods vidually in running sea water. Since different experiments were conducted on different days, crabs were never tested to the Study site same conditions if they were recollected, but may be used in the other experiments of different conditions. Field work took place on a mangrove site located 17 km south In each experimental condition, 30 individual hermit crabs, of Safaga city (26210N and 33480E). The site represents a without a shell, were used. Crabs were found to increase their mangrove swamp protected area that is monitored by the orientation behavior activity if they are removed from their Nature Conservation Sector at the Egyptian Environmental shells (Diaz et al., 2001). The test was initiated by placing crabs Affairs Agency (NCS/EEAA) and characterized by a high individually within a short polyvinylchloride cylinder (PVC) diversity of marine life. The areas near the shore have mono- (4 cm height, 5 cm diameter) in the center of the arena. The specific forests of mangrove trees (Avicennia marina) that are individual hermit crab was removed by hand from its holding intersected by channels of different sizes. At this site, the arena and this handling caused the hermit crab to expose Effects of visual and chemical cues on orientation behavior of the Red Sea hermit crab Clibanarius signatus 97 briefly to air (less than 2 s) which may have an effect on its caeruleum (Sowerby, 1855), Nassarius arcularius (Ro¨ ding, behavior during the experiment. Therefore, the individual her- 1798), Nerita albicilla (Linnaeus, 1758), Planaxis sulcatus mit crab was left for 3 min acclimation period, then the PVC (Born, 1778) and Strombus mutabilis (Swainson, 1821) cylinder was gently lifted and the crab movement direction (Sharabati, 1984). Before the experiment was conducted, the was observed and recorded. The first point of contact with are- selected shells were heated to 200 C in a muffle furnace for na wall having a radius equal to the distance from the center of 4 h to eliminate organic odors and to give them a uniform gray the arena to the target was recorded as the orientation direc- appearance (Diaz et al., 1995a). Each shell was positioned in- tion. Crabs that did not reach the arena wall within 60 s were side the translucent arena and thus appeared as a gray shape recorded as not responding. Flat black rectangles and empty against a white background and never rise above water level. shells were used as targets and subtended at fixed angles as Shells were presented in four possible visual positions; (1) ante- viewed from the center of the arena and served as visual cues. rior, with anterior end of shell toward crab and aperture facing To avoid biasing the direction of orientation, the direction of down; (2) lateral, with aperture facing down; (3) upside-down, the crab was systematically rotated 90 clockwise for each with aperture facing up and (4) posterior with apical whorls to- experimental trial. ward crab and aperture facing down (Fig. 1). The anterior and In trials of Experiment I, hermit crabs were used to test vi- posterior positions of N. albicilla had similar silhouettes, and sual orientation toward two flat black cardboard rectangles of thus only posterior position was tested. 3 cm height that attached separately to the inside wall of arena In trials of Experiments III and IV, hermit crabs were ex- subtended at angles of 20 and 150 (±10 accuracy). posed to two types of chemical cues that were prepared in In trials of Experiment II, crab orientation was subse- clean sea water. Calcium chloride (CaCl2) was used as a source quently tested in the presence of different silhouettes of six gas- for calcium (Mesce, 1982). Four calcium concentrations were tropod shell species subtended at a fixed angle; 20 (±10 used in the present investigation, 10, 20, 40 and 80 mg/l. These accuracy), as observed from the center of the arena. The used concentrations were added to the levels of calcium and chlo- shell species were Bursa granularis (Ro¨ ding, 1798), Cerithium ride in ambient sea water. Calcium concentration in the sea

Anterior Lateral Upside-Down Posterior

Bursa granularis

Cerithium caeruleum

Nassarius arcularius

Nerita albicilla

Planaxis sulcatus

Strombus mutabilis

Figure 1 Gastropod shells used as targets in the present investigation. Species names are shown at the bottom of each row. Positions of shells as viewed from the center of the arena are indicated at the top of each column. 98 Tarek Gad El-Kareem Ismail water of studied site was analyzed using flame photometer Table 1 Orientation of C. signatus to a solid target located at (JENWAY PFP7), and its value was 468 mg/l. two different angles. Each circular solid point represents The crab Metopograpsus sp. was used as the crab predator. directional response of one hermit crab. The control (A) shows Many individuals of this species are present in the investigated orientation in the white background sea water. N, number of C. site hiding among mangrove roots and surrounded by crashed signatus tested; n, number of C. signatus responding; Z, value of shells. Predator odor concentration was developed by main- Rayleigh test; U, value of V-test; A%, percentage of crab taining a single predator crab of known weight in clean ambi- attracted to the target; Fig., figure showing orientation distri- ent sea water for different lengths of time. Crab predator odor bution; ns, not significant. *P < 0.05, **P < 0.01, ***P 6 0.001. was in units of grams of crab/l/h. Three predator odor concentrations were used 1, 2 and 4 g/l/h. To insure the continued Angle Nn Z u A% Fig. effectiveness of chemical cues, the sea water and chemical cues Control – 30 22 0.28ns ––1A were renewed every 30 min, since chemical cues are biologi- Target 1 20 30 23 2.68* 2.18** 52.17 1B cally consumed over time (Rittschof et al., 1984). Target 2 150 30 25 7.7 3.12** 52.00 1C In Experiment V, eyes of individual hermit crabs were blinded using nail polish and test orientation to a fixed shell target in the presence of pervious two chemical cues. Chemical a single target subtended at 20 and 150 (±10 accuracy) in cues were prepared as in Experiments III and IV. The hermit separate trials, individuals of C. signatus exhibited significant crabs were left for 2 h before beginning of the experiment for orientation toward each of these targets (Table 1 and acclimation. Fig. 2B, C). The attraction percentage of hermit crabs to each The movement with surrounding white wall of the arena target was 52.17% and 52%, respectively. alone was tested as a control for orientation in the absence In clean sea water trials of Experiment II, an average of of apparent visual cues (absence of a shell) with or without 92.27% of the tested hermit crabs responded within the 60 s two previous chemical cues. trial interval (Table 2). When individuals of C. signatus were presented with different views of six shell species, they were Statistical analysis not attracted to shells of B. granularis, N. arcularius and N. albicilla in any position (Table 2 and Fig. 3A–D, I–P). C. caeruleum and S. mutabilis were attractive only in the lateral and Directional orientation of responding hermit crabs was deter- upside-down positions recording the highest attraction percent- mined by the circular statistical methods (Zar, 1999) using Ori- age (lateral: 46.7%, 60.7%; upside-down: 53.3%, 53.6) (Table 2 ana program. The mean angle and the mean vector length were and Fig. 3F–G, V–W), while P. sulcatus was attractive in the calculated for each experimental condition, while the expected upside-down position only recording the lowest attraction per- angle was established as the middle of target and as the oppo- centage (21.4%) (Table 2 and Fig. 3S). Although Rayleigh test site to the target in case of predator odor. (Z) showed aggregation orientation (P < 0.05) with some posi- There were two statistical tests for directional orientation. tions of B. granularis (posterior), N. arcularius (lateral and up- First, the Rayleigh test (Z) determined whether the orientation side-down), N. albicilla (posterior), P. sulcatus (anterior) and S. distribution was significantly different from uniformity. Sec- mutabilis (posterior), the u value was not significant for them ond, the (V) test calculated to determine whether the distribu- because it tested for orientation toward the target, which means tion was different from uniformity by using both the mean that individual hermit crabs did not orient toward the target angle and an expected mean angle. If the (Z) statistic indicated shells. Because the hermit crab individuals recorded the highest that the distribution was non-uniform (P < 0.05), then the (V) attraction percentage with upside-down position of both C. test was run and (u) value indicated the significance level. caeruleum and S. mutabilis shell types, these shells and their up- Although, the Rayleigh test could indicate the presence of a side-down position were used in the next experiments to evalu- non-uniform distribution, the (V) test might be insignificant ate the effect of chemical cue concentrations. because the mean vector was not oriented toward the target. In addition for each distribution, the proportion of the Chemical cues responding hermit crabs and those attracted to the target (A%) was calculated. The proportions of attraction were com- pared to each other by means of a multiple comparisons test In Experiment III, the calcium cues were added to the ambient for proportions (Zar, 1999). sea water. An average of 82.59% of the tested hermit crabs responded within the 60 s trial interval (Table 3). In white background arena (sea water containing 10 mg/l calcium, Results representing control trial, no visual cues), individuals of C. signatus had uniform orientation distribution (Table 3 and No chemical cues Fig. 4A). When exposed to different concentrations of calcium cues in the presence of two target shells subtended at 20 In clean sea water trials of Experiment I, an average of 93.19% (±10 accuracy), the percentage of attraction increased as cal- of the tested hermit crabs responded within the 60 s trial inter- cium concentration increased from 10 to 40 mg/l and slightly val (Table 1). When the arena was surrounded by the white decreased with 80 mg/l for both two shell types (Table 3 and background alone (control, no visual cues), the orientation Fig. 4B–I). However, this increase in attraction percentage of C. signatus individuals was not aggregated in any direction was insignificant (multiple comparisons test, P>0.05). and they showed a uniform distribution (Rayleigh test, In Experiment IV, the individuals of hermit crab were pre- P > 0.05) (Table 1 and Fig. 2A). This indicates that there sented with predator odor. In white background arena (sea was no bias due to the used technique. When presented with water containing 1 g/l/h predator cue, representing control Effects of visual and chemical cues on orientation behavior of the Red Sea hermit crab Clibanarius signatus 99

Control Angle 20º Angle 150 º 0 0 0 A B C

270 90 270 90 270 90

180 180 180

Figure 2 Orientation of C. signatus to 20 and 150 (±10 accuracy) targets. Solid dots represent the directional response of each hermit crab. Black rhombus symbol outside each circle refers to the direction of the target.

Table 2 Orientation of C. signatus to six shell species subtended at 20 (±10 accuracy) presented in different views in white background sea water. Symbols as Table 1. Shell Position Nn Z u A% Fig. Bursa granularis Anterior 30 0 1.66ns – 6.6 2A Lateral 30 30 0.98ns – 3.3 2B Upside-down 30 27 0.35ns –02C Posterior 30 24 6.36* 1.32ns 0 2D Cerithium caeruleum Anterior 30 27 1.6ns –02E Lateral 30 30 9.15** 4.03** 46.7 2F Upside-down 30 30 6.22* 3.45** 53.3 2G Posterior 30 26 0.52ns – 15.8 2H Nassarius arcularius Anterior 30 25 1.7ns –02I Lateral 30 24 11.4** 0.28ns 0 2J Upside-down 30 28 5.1* 1.46ns 0 2K Posterior 30 26 1.6ns –02L Nerita albicilla Anterior 30 29 2.1ns – 3.45 22M Lateral 30 29 0.34ns – 10.3 2N Upside-down 30 29 0.91ns – 13.8 2O Posterior 30 27 3.85* 2.45ns 14.8 2P Planaxis sulcatus Anterior 30 30 4.18* 2.19ns 0 2Q Lateral 30 25 0.24ns –82R Upside-down 30 28 2.98* 2.1* 21.4 2S Posterior 30 29 1.58ns – 6.89 2T Strombus mutabilis Anterior 30 29 0.97ns – 31.0 2U Lateral 30 28 5.82** 3.17** 53.6 2V Upside-down 30 28 6.1** 2.83** 60.7 2W Posterior 30 30 5.1** 1.23ns 16.7 2X trial, no visual cues), individuals of C. signatus had uniform ori- crabs move in circles around the arena center or just stopped entation distribution (Table 4 and Fig. 5A). When individuals moving. According to the present results (Table 2), the S. mut- of C. signatus presented with predator odor concentrations of abilis was used in the present experiment and subtended at 20 1, 2 and 4 g/l/h in the presence of two target shells subtended (±10 accuracy). When calcium cue was added to sea water at 20 (±10 accuracy), their attraction percentage showed (only two concentrations were used, 40 and 80 mg/l), the per- insignificant decrease from 1 to 4 g/l/h (multiple comparisons centage of responding decreased to 66.7% and 60.0%, respec- test, P>0.05) (Table 4 and Fig. 5B–G). However, two re- tively (Table 5 and Fig. 6B, C). With the predator odor sponses appeared with predator odor. Firstly, the hermit crabs concentrations (1, 2 and 4 g/l/h), the responding percentage oriented away from the 20 ±10 target, and secondly they ori- strongly declined recording 36.7%, 20.0% and 26.7%, respec- ented significantly toward the opposite direction of the arena. tively (Table 5 and Fig. 6D–F). Hermit crab individuals showed random orientation (Rayleigh test, P > 0.05) in all tri- Hermit crabs with blinded-eyes als of the two chemical cues and the (u) value indicated no orientation toward the target. In Experiment V, the eyes of the hermit crab individuals were blinded using nail polish. In white background arena with Discussion clean sea water (control, no chemical cues, no visual cues) only 23 (76.7%) individuals reached the wall of the arena (Table 5 Chemical cues reflect the characteristic of different habitats and Fig. 6A). The responding individual crabs moved slowly in and they can modify visual orientation of many crustaceans the arena until touching the wall, while the non-responding (Diaz et al., 1999; Chiussi et al., 2001). 100 Tarek Gad El-Kareem Ismail

Anterior Lateral Upside-Down Posterior 0 0 0 0 A B C D

270 90 270 90 270 90 270 90

180 180 180 180 Bursa granularis 0 0 0 0 E F G H

270 90 270 90 270 90 270 90

180 180 180 180 Cerithium caeruleum 0 0 0 0 I J K L

270 90 270 90 270 90 270 90

180 180 180 180 Nassarius arcularius 0 0 0 0 M N O P

270 90 270 90 270 90 270 90

180 180 180 180 Nerita albicilla 0 0 0 0 Q R S T

270 90 270 90 270 90 270 90

180 180 180 180 Planaxis sulcatus 0 0 0 0 U V W X

270 90 270 90 270 90 270 90

180 180 180 180 Strombus mutabilis

Figure 3 Orientation of C. signatus to 20 (±10 accuracy) target of varied shell types in different views. Solid dots represent the directional response of each hermit crab. Black rhombus symbol outside each circle refers to the direction of the target.

The first aim of the present work was that the hermit crab as shell seeking behavior. Similar results were obtained for C. signatus has the capability to use visual cues for orientation. other relative tropical hermit crabs as Clibanarius antillensis This hypothesis was verified as the individual hermit crabs ori- (Chiussi et al., 2001) and Clibanarius vittatus (Hazlett, 1982; ented toward the 20 and 150 black rectangle targets in clean Diaz et al., 1995b). This also gave an indication that the crab sea water. Since the present individual hermit crabs were tested response is not a chemically induced visual orientation toward without their shells, this visual directional orientation proba- black cardboard rectangles. bly reflects the demands for occupation of shells or perhaps The second aim was that C. signatus has the ability to dif- seeking a shelter (as a rock). This behavior can be considered ferentiate between different targets based on visual informa- Effects of visual and chemical cues on orientation behavior of the Red Sea hermit crab Clibanarius signatus 101

Table 3 Orientation of C. signatus to different shell species subtended at 20 (±10 accuracy) presented in upside-down position in calcium odor. Symbols as Table 1. Shell Position Nn Ca2+ conc. (mg) Zu A% Fig. Control 30 20 10 0.37ns ––3A Cerithium caeruleum Upside-down 30 24 10 3.16* 2.49* 45.8 3B 30 27 20 5.1* 3.16** 59.3 3C 30 26 40 7.82*** 3.95*** 73.1 3D 30 25 80 8.95*** 3.24*** 72.0 3E Strombus mutabilis Upside-down 30 25 10 3.78* 2.35* 56.0 3F 30 26 20 6.36** 3.38*** 61.5 3G 30 24 40 11.3** 4.64*** 66.7 3H 30 26 80 15.2*** 5.48*** 65.4 3I

0 A

270 90

10mg/l 180 Control 0 0 0 0 B C D E

270 90 270 90 270 90 270 90

10mg/l 180 20mg/l 180 40mg/l 180 80mg/l 180 Cerithium caeruleum 0 0 0 0 F G H I

270 90 270 90 270 90 270 90

40mg/l 80mg/l 10mg/l 180 20mg/l 180 180 180 Strombus mutabilis

Figure 4 Orientation of C. signatus to 20 (±10 accuracy) target presented in upside-down position of shells Cerithium caeruleum and Strombus mutabilis in water with different calcium cue concentrations. Black rhombus symbol outside each circle refers to the direction of the target. tion. This was confirmed by the attraction of hermit crab indi- and other decapods as fiddler crab Uca pugilator (Herrnkind, viduals not only to specific shell types but also to specific posi- 1983; Langdon and Herrnkind, 1985). tions of these shells, with the greatest attractiveness to C. The lateral and upside-down positions of both C. caeruleum caeruleum and S. mutabilis in lateral and upside-down posi- and S. mutabilis mimic the diamond shape. While, non-attrac- tions. Also, the hermit crab individuals were not maximally at- tive positions of tested shells resemble a square (N. arcularius, tracted to the tallest (B. granularis) or shortest (N. albicilla) anterior; N. albicilla, lateral; P. sulcatus, posterior; S. mutabi- shell, or to the widest (B. granularis) or narrowest (P. sulcatus) lis, anterior), a semicircle (B. granularis, anterior; C. caeruleum, shell aperture. This suggests that the orientation is not clearly anterior, posterior; N. albicilla, posterior; P. sulcatus, ante- related to simple dimension characteristics of the shell (height rior), a circle (B. granularis, posterior; S. mutabilis, posterior) and width) and the attraction must be based on shape recogni- and a rectangle (B. granularis, lateral, upside-down; N. arcula- tion (Chiussi, 2002). In contrast to the present result, hermit rius, lateral, upside-down; N. albicilla, upside-down; P. sulca- crab C. vittatus was found to use shell dimensions in its direc- tus, lateral, upside-down). Although, these relationships tional orientation (Diaz et al., 1994). Generally, visual shape suggested that individuals of C. signatus were attracted simply discrimination has been observed in hermit crab C. vittatus to specific shapes, more complex shell recognition must be in- (Hazlett, 1982; Orihuela et al., 1992; Diaz et al., 1994, 1995a) volved because crabs were not attracted to N. arcularius in any 102 Tarek Gad El-Kareem Ismail

Table 4 Orientation of C. signatus to two shell species subtended at 20 (±10 accuracy) presented in upside-down position in predator odor. Symbols as Table 1; Pred. od, predator odor. Shell Position Nn Pred. od. (g/l/h) Zu A% Fig. Control 30 17 1 0.48ns ––4A Cerithium caeruleum Upside-down 30 22 1 4.75* 3.0ns 18.2 4B 30 20 2 7.64** 3.6ns 10.0 4C 30 23 4 0.65ns – 8.7 4D Strombus mutabilis Upside-down 30 24 1 3.29* 2.6ns 20.8 4E 30 19 2 3.37* 2.3ns 15.8 4F 30 21 4 0.78ns – 9.5 4G

Figure 5 Orientation of C. signatus to 20 (±10 accuracy) target presented in upside-down position of shells Cerithium caeruleum and Strombus mutabilis in water with different predator odor concentrations. Black rhombus symbol outside each circle refers to the direction of the target.

Table 5 Orientation of blinded C. signatus to shell of Strombus mutabilis subtended at 20 (±10 accuracy) in different concentrations of calcium and predator cues. Symbols as Table 1; Conc., concentration. Shell Cue Nn Conc. ZuA% Fig. Control – 30 23 – 0.52ns –– 5A Strombus mutabilis Calcium 30 20 40 mg 1.66ns –5 5B 30 18 80 mg 0.99ns –0 5C Predator 30 11 1 g/l/h 0.05ns – 9.1 5D 30 6 2 g/l/h 0.66ns –0 5E 30 8 4 g/l/h 0.65ns –0 5F

positions. Diaz et al. (1994, 1995a) suggested that attraction to avoided by hermit crab C. signatus. This can be interpreted speciﬁc shapes may be related to the potential habitability of as an escape response from predators that mimic these posi- shells presented by these shapes. Ismail (2010) illustrated that tions (Diaz et al., 1994, 2001) or that these positions did not hermit crab C. signatus preferred S. mutabilis and C. caeruleum reﬂect the preferred shell type (Diaz et al., 1995a). Such visu- gastropod shells over other abundant shell types. Also, the re- ally mediated predator avoidance responses have been ob- sults refer to those within a species, a shell may (Fig. 2F, G, V served in other decapods (Orihuela et al., 1992; Diaz et al., and W) or may not (Fig. 2E, H, U and X) be distinguished in 1994, 1995b, 1999, 2003). Thus, C. signatus exhibits similar different positions. visually mediated predator avoidance responses. The non-attractive positions of the tested shells which The third goal was that C. signatus can change its behavior resemble a quadrate, circle, semicircle and rectangle were based on visual and chemical cues. This was achieved in two Effects of visual and chemical cues on orientation behavior of the Red Sea hermit crab Clibanarius signatus 103

0 A

270 90

180 Control 0 0 B C

270 90 270 90

180 180 Ca2+ 40mg Ca2+ 80mg 0 0 0 D E F

270 90 270 90 270 90

180 180 180 Predator 1g/l/h Predator 2g/l/h Predator 4g/l/h

Figure 6 Orientation of C. signatus to 20 (±10 accuracy) target of varied shell types in different views. Solid dots represent the directional response of each crab. Black rhombus symbol outside each circle refers to the direction of the target. steps, first, the ability of crabs to orient toward the two shell The second step was the effect of predation odor on orien- targets in the presence of calcium cue. When exposed to white tation of C. signatus toward the shell targets. Upon exposure background, tested individuals showed a uniform circular dis- to the 20 two shell targets and predator cues, some individual tribution. These results indicate that there were no uncon- hermit crabs reversed the orientation as they swim away from trolled cues in the test arena. Upon exposure to the 20 the target toward the opposite direction. This response may be target and calcium concentrations of 10–40 mg/l, responding representing predator avoidance response or shelter seeking and orientation of C. signatus individuals to shell targets loca- behavior (Rotjan et al., 2004; Mima et al., 2003; Diaz et al., tion increased up to a maximum attraction percentage and 1999). Huang et al. (2005) reported that in the light, individu- then attraction became stable when crabs were exposed to als of the shrimp Synalpheus demani are highly responsive and higher calcium concentration (80 mg/l). Thus, orientation to orient to visual targets, regardless the presence of odor sug- fixed visual cues (shell targets) changed with chemical concen- gesting that light and vision are the main drivers of S. demani tration. Calcium is used as a chemical cue for identification of orientation. gastropod shells by many hermit crab species as Pagurus sam- Predator avoidance responses were interpreted as either uelis, Pagurus hirsutiusculus (Mesce, 1982) and C. antillensis alarm (fright) or escape responses. Alarm reactions consisted (Chiussi et al., 2001). Moreover, sensitivity to calcium may of either remaining immobile or swimming in all directions, enable some hermit crab species to locate particularly buried whereas swimming away from a target was considered an es- shells which increase the number of shells available for habi- cape response (Atema and Burd, 1975; Lima and Dill, 1990; tation (Chiussi et al., 2001). It is known that living gastropods Rahman et al., 2000). Escape responses are known to be found secrete periostracum around their shells which reduces the among decapods such as hermit crabs C. vittatus and P. sam- amount of calcium lost from shell surface (Mesce, 1982). uelis (Orihuela et al., 1992; Billock and Dunbar, 2009), crab But after predation or death, calcium is released from the Chasmagnathus granulatus (Romano et al., 1990), and Calli- shells and its cue oriented hermit crab individuals toward gas- nectes sapidus (Woodbury, 1986). tropod predation or death sites (McLean, 1974; Rittschof In general, hermit crabs respond differently according to et al., 1990). Once hermit crab individuals reached these sites, different stimuli in their environment. They increase the rate visual cues may be used for location of shells (Ohta, 1971; of locomotion in the presence of shell-breaking crabs (Hazlett, McLean, 1974; Rittschof, 1980a). However, some hermit 1996; Rittschof and Hazlett, 1997) while they decrease the rate crabs as P. samuelis do not rely on calcium cue in selecting of locomotion in the presence of visual predators (Hazlett, a shell (Reese, 1969). On the other hand, Mesce (1982) re- 1997), or alter their orientation response to visual cues about ported that when seawater became saturated with calcium, potential refuges (Chiussi et al., 2001). it masked the calcium cues of the shell, leading to uniform It is known that the blind hermit crabs relay on chemore- orientation distribution. ceptors for extraction of directional information from a 104 Tarek Gad El-Kareem Ismail chemical stimulus in a surrounding environment (Hazlett, Chiussi, R., 2002. Orientation and shape discrimination in juveniles 1975). This result confirmed by the present result, in which and adults of the mangrove crab Aratus pisonii (H. Milne Edwards, the blind hermit crab C. signatus exposed to 20 shell target 1837): effect of predator and chemical cues. Marine and Freshwater combined with either calcium or predator cues, exhibited uni- Behavior and Physiology 36, 41–50. Chiussi, R., Diaz, H., Rittschof, D., Forward, R., 2001. Orientation of form orientation distribution. The responsiveness was higher the hermit crab Clibanarius antillensis: effects of visual and with calcium cues than predator cues. The majority of blinded chemical cues. Journal of Crustacean Biology 21 (3), 593–605. hermit crab individuals when exposed to predator cues exhibit Chivers, D.P., Smith, R.J.F., 1998. Chemical alarm signaling in stationary behavior or moving in circles around the arena cen- aquatic predator–prey systems: a review and prospectus. Eco- ter. These results suggest that C. signatus are able to detect by science, 5338–5352. odor the potential presence of its natural predators. Although Diaz, H., Forward Jr., R.B., Orihuela, B., Rittschof, D., 1994. stationary behavior is not a direct antipredator response, Chemically stimulated visual orientation and shape discrimination which could put the individuals in a position more vulnerable by the hermit crab Clibanarius vittatus (Bosc). Journal of Crusta- to predation, it demonstrates that tested hermit crabs can de- cean Biology 14, 20–26. tect and respond to chemical signal using chemoreceptors Diaz, H., Orihuela, B., Rittschof, D., Forward Jr., .R.B., 1995a. Visual orientation to gastropod shells by chemically stimulated hermit (Rittschof et al., 1992; Iglesias, 2007). Thus in the absence of crabs, Clibanarius vittatus (Bosc). Journal of Crustacean Biology vision, individual hermit crabs were able to detect both cal- 15, 70–78. cium and predator cues and have different responses regarding Diaz, H., Orihuela, B., Rittschof, D., Forward Jr., R.B., 1995b. Visual them. It can be concluded that vision operates as the primary orientation of postlarvae and juvenile mangrove crabs. Journal of or more reliable cue to use for orientation. However, olfaction Crustacean Biology 15, 671–678. operates as secondary cue. Once vision is absent olfaction be- Diaz, H., Orihuela, B., Forward Jr., R.B., Rittschof, D., 1999. comes the primary one. In this respect, Moore and Bergman Orientation of blue crab, Callinectes sapidus (Rathbun), megalopae: (2005) reported that olfactory signals become more important responses to visual and chemical cues. Journal of Experimental when vision is scrawny due to environmental conditions such Marine Biology and Ecology 233, 25–40. as water clarity or darkness. Diaz, H., Orihuela, B., Forward Jr., R.B., Rittschof, D., 2001. Effects of chemical cues on visual orientation of juvenile blue crabs, Generally, when individuals of C. signatus are faced with Callinectes sapidus (Rathbun). Journal of Experimental Marine the chemical cue of a predator, they have two typical antipred- Biology and Ecology 266, 1–15. ator behaviors: fleeing from the area or stop moving. These re- Diaz, H., Orihuela, B., Forward Jr., R.B., Rittschof, D., 2003. sults clearly indicated that the chemical cues altered behavior Orientation of juvenile blue crabs, Callinectes sapidus (Rathbun), to and induced alarm responses in which the crabs either re- currents, chemicals, and visual cues. Journal Crustacean Biology 23 mained motionless or swam in all directions (Scarratt and (1), 15–22. Godin, 1992; Mima et al., 2003). A turn-and-move behavior Dicke, M., Grostal, P., 2001. Chemical detection of natural enemies by presumably assists in acquiring information as a scanning arthropods: an ecological perspective. Annual Review of Ecology behavior. and Systematics 32, 1–23. Finally, it can be concluded that hermit crab C. signatus has Dill, L.M., 1987. 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