Movement and aggregation in the fluted giant clam (Tridacna squamosa L.)

Journal of Experimental Marine Biology and Ecology 342 (2007) 269 – 281 www.elsevier.com/locate/jembe

Movement and aggregation in the fluted giant clam (Tridacna squamosa L.) Danwei Huang a,⁎, Peter A. Todd a , James R. Guest b a

Marine Biology Laboratory, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543, Singapore b School of Biology and Psychology, Division of Biology, The Ridley Building, Newcastle University, Newcastle upon Tyne, NE17RU, United Kingdom Received 15 June 2006; received in revised form 12 September 2006; accepted 30 October 2006

Abstract Aquaculture has been the traditional focus of tridacnid giant clam research whereas their ecology and behaviour have received much less attention. This study was based on the observation that juvenile fluted giant clams (Tridacna squamosa), when evenly distributed in a tank, will move and aggregate over time. We observed movement in clams ranging from 10 to 313 mm in shell length and ‘climbing’ up the sides of tanks was noted for clams with lengths between 10 and 22 mm. Locomotion also occurred after byssal attachment to the substrate; there was a highly significant association between type of movement (i.e. translation, rotation and no movement) and presence of attachment. Tests for phototaxis were negative. Aggregation was examined by placing clams in regular patterns on grids. After three days in the aquarium and 24 h in the field, their positions were analysed to obtain a statistical parameter for ‘clumpiness’. This was found to be greater in the live clam runs than both random walk and random distribution simulations, suggesting that clams were attracted to conspecifics. The latter was tested by recording clam movement with respect to five types of fixed ‘targets’ (i.e. live clam, fouled clam shell, foul-free clam shell, random inanimate object and none). The test clams moved, nonsignificantly, towards live clam targets and displayed higher mobility compared to tests with other target objects; a negative correlation between mobility and clam length was also observed. A choice experiment using bidirectional water inflow with clam effluent as one source resulted in clams moving toward the effluent, offering the first direct support for positive chemotaxis among conspecifics in Bivalvia. Together, our results indicate the presence of chemical signalling among clams, leading to movement toward one another and clumping. Aggregation could serve several ecological functions, such as defence against predation, physical stabilisation and facilitation of reproduction. With worldwide decline in natural giant clam densities, the opportunity for conspecific clumping is reduced, and local stocks could be facing increased vulnerability to Allee affects. © 2006 Elsevier B.V. All rights reserved. Keywords: Aggregation; Behaviour; Chemotaxis; Giant clam; Movement; Tridacna

1. Introduction Giant clams (Bivalvia: Tridacnidae) are highly specialised bivalve molluscs that inhabit shallow waters of tropical Indo-Pacific coral reefs (Rosewater, 1965). Re⁎ Corresponding author. Tel.: +65 68746867; fax: +65 67792486. E-mail address: [email protected] (D. Huang). 0022-0981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2006.10.051

search has focused primarily on the aquaculture of giant clams (e.g. Beckvar, 1981; Hart et al., 1998) and associated themes such as physiology (e.g. Leggat et al., 2003), stemming from their long-established role as a traditional food source for coastal peoples in the region (Munro and Heslinga, 1983; Alcala, 1986; Braley, 1992; Lucas, 1994). There is, however, a paucity of information on the ecology of giant clams (but see Stasek, 1965; Dolgov, 1991; Belda-

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Baillie et al., 1999), and fundamental questions regarding their behaviour have not been examined in detail. Hence, this study focuses on movement and aggregation in the fluted giant clam, Tridacna squamosa. Locomotion is crucial for the survival of bivalves (Tan, 1975; Amyot and Downing, 1997; Waller et al., 1999). Yet, save for anecdotal observations recorded in a seminal paper by Yonge (1936) and a short description of early juveniles (Reid et al., 1992), this behaviour is not well-documented in tridacnid clams. Conversely, locomotion in other bivalve taxa has been examined since the 19th century (e.g. Lockwood, 1870) and a range of movements have been documented. For instance, swimming, typically involving the rapid adduction of valves, has been described in several scallop species (family Pectinidae) (e.g. Moore and Trueman, 1971; Morton, 1980; Brand, 1991), and in the genera Lima (Limidae) (Gilmour, 1967) and Ensis (Pharidae) (Drew, 1907). Thread drifting, a form of pelagic translocation using foot-produced filamentous threads, was reported in some bivalve molluscs and is a key mechanism of post-settlement dispersal (Sigurdsson et al., 1976; Lane et al., 1985; Ackerman et al., 1994). Particularly, bivalves are exemplified by the large number of taxa known to burrow into soft substratum (Trueman, 1966, 1967, 1968a,b). The mechanism of this action is similar to movement along the surface of a substratum, generally achieved by projection of the foot to anchor onto it and then drawing the animal towards the intended direction of motion (Trueman, 1983; Davenport, 1988), a process that is then repeated. An extension of this behaviour is leaping, where the foot lifts the shell off the substrate and in the direction of movement (e.g. Ansell, 1967, 1969). Giant clams are also known to be able to move along the substratum and, with the aid of byssal threads, climb up vertical surfaces (Yonge, 1936; Reid et al., 1992); but this behaviour has not been quantified. Bivalves may orientate and move according to several abiotic factors (e.g. Akberali and Davenport, 1982; Maguire et al., 1999; Waller et al., 1999), including light and/or gravity (Morton, 1960, 1962; Uryu et al., 1996). In Montacuta ferruginosa, for instance, negative phototaxis and positive geotropism amplify a chemotactic response that serves to move the commensal down towards its host urchin (Morton, 1962). Several workers have observed physical reactions of giant clams to light stimuli, including withdrawal of the mantle with sudden light dimming (Land, 2003) and active orientation of the mantle tissue in response to increased light intensity (Wilkens, 1986). The most spectacular response recorded is probably the accurate squirting of water from the exhalent siphon of T. maxima towards nearby ob-

jects (Stasek, 1965; McMichael, 1974). This ability is particularly surprising given that the giant clam's visual system is relatively simple, comprising hundreds of pinhole eyes on the mantle edges (Fankboner, 1981; Wilkens, 1984). Since mobile symbiotic zooxanthellae have been known to actively move toward green illuminated areas (Hollingsworth et al., 2005), our poor understanding of the host's movement in response to light, and of clam motility in general, is surprising. Aggregation between conspecifics is one of the possible outcomes of movement and is a major determinant of survival in animals (Allee, 1931; Turchin, 1989). It is a key mediator of spatial disposition of ecological resources, and potentially shapes predator–prey and conspecific interactions (Taylor and Taylor, 1977; Turchin, 1989; Cummings et al., 1997). The aggregative behaviour of several bivalve species has been investigated, and although tridacnid clams were observed to display such a tendency (McMichael, 1974; Yamaguchi, 1977), no studies have examined underlying mechanisms. Two possible proximate causes of this behaviour in bivalves have been suggested. First, clumping could be a result of individuals moving randomly, stopping when groups are formed (e.g. Senawong, 1970; Uryu et al., 1996). Second, gregariousness among individuals, especially chemotactic attraction, may result in movement toward one another and the formation of dense aggregations (Morton, 1960; Côté and Jelnikar, 1999). Direct tests of chemotaxis are rare, mainly due to the difficulty of detecting the effect in aquarium tanks (Uryu et al., 1996). As mentioned, M. ferruginosa was shown to move towards its urchin host by chemical signaling (Morton, 1962), but no studies have examined possible chemotactic response of bivalves in the presence of conspecifics. The purpose of this study is to examine the following hypotheses: (i) clam movement is affected by byssal attachment and/or light; (ii) aggregation of clams occur in aquarium tanks and in the field; and (iii) clams are attracted to one another and exhibit positive intraspecific chemotaxis. As there is currently little understanding of giant clam behaviour, findings from this study will provide important baseline information regarding their movement and aggregative patterns. Results may also be useful for the development of clam restoration techniques and mariculture strategies. 2. Materials and methods 2.1. General experimental procedures This study was carried out at the marine aquaculture facility in the Tropical Marine Science Institute (TMSI)

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on St John's Island, Singapore, from July 2005 to March 2006. Test organisms were a single cohort of juvenile giant clams (shell length 23–63 mm) produced from the spawning of T. squamosa brood stock in March 2004. Large circular tanks (diameter 1.1 m and height 0.7 m) were used for all aquarium experiments (unless otherwise stated), filled to the 50 cm mark with filtered water in a flow-through system. The water system and air supply were switched off during the experiments to minimise water motion. As the bottom surfaces were not even, unless stated otherwise, acrylic sheets (diameter 1.05 m and thickness 8 mm) supported by cement blocks were placed horizontally (tested with a spirit level) in the tanks. Sheets were roughened with a random orbital sander fitted with 40-grade sandpaper. 2.2. Clam movement and phototaxis T. squamosa movement was examined in clams of various sizes, ranging from juveniles ∼ 10 mm in shell length to adults N 300 mm. Time-lapse photography was also used to investigate the mechanism of movement from various views. Dissections were made to examine anatomical features involved in the behaviour. To examine how byssal attachment affects clam movement, the number of juvenile clams exhibiting two types of movement – rotation and translation – were recorded. Rotation was defined as change of clam orientation without the approximate centre of the clam being shifted from its initial position, while translation was defined as lateral movement away from the original position. A total of 120 clams (shell length 30–52 mm) were placed on the bottoms of four circular tanks in groups of 30, approximately evenly spaced apart (∼ 100 mm), and a control was set up with another 120 clams placed in four tanks on smooth acrylic surfaces

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where byssal attachment was impossible (as determined in pilot studies). Movement was restrained by placing a PVC ring (diameter 68 mm) around each animal. When all clams in the first treatment were firmly anchored to the tank bottoms, the acrylic sheets in the control treatment and all PVC rings were removed slowly and carefully with minimal disturbance. Lines were penciled on the bases of the tanks to mark initial positions of the clams, and movement data were collected after seven days. Pearson Chi-square test was applied on the 3 × 2 frequency table (‘translation’, ‘rotation’ and ‘no movement’ vs ‘byssally attached’ and ‘unattached’). We investigated giant clam's locomotive response to light by placing individual juvenile clams (shell length 32–52 mm) at the center of clear rectangular plastic tanks (length 27 cm × width 16 cm × height 18 cm) in haphazard orientations (n = 40). Three light treatments were set up using wide-spectrum SYLVANIA F36/ GRO fluorescent tubes (see Harriss et al., 1970; O'Brien et al., 1984), i.e. (i) light from one direction of the clam, A; (ii) light from two opposite directions, A and B; and (iii) no light. Light sources were placed 30 cm away from, and 15 cm above, the experimental subject to ensure a distinct decreasing gradient of light intensity from the tank periphery to its center (Fig. 1). Final positions of the clams after 3 h were recorded as having moved towards direction A, B, or remaining at the centre (neutral). Pearson Chi-square test was performed on the 3 × 3 frequency table (‘towards A’, ‘neutral’ and ‘towards B’ vs light source direction ‘A’, ‘A and B’ and ‘no light’). 2.3. Aggregation in giant clams We tested for possible aggregation of clams by analysing their distribution over time on roughened

Fig. 1. Diagram showing experimental setup to evaluate phototaxis of clams. Illustrated here is the treatment ‘light from one direction’.

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acrylic sheets in the circular aquarium tanks. Thirty-six clams (shell length 25–63 mm) were placed 10 cm apart in a regular pattern on a 60 × 60 cm2 grid, consisting of 30 × 30 4 cm2 cells drawn on the acrylic sheet using a permanent ink marker (n = 6; total 216 clams). Cartesian coordinates of the clams' final positions were recorded after three days and spatial analysis was performed to determine the degree of aggregation by obtaining an index for ‘clumpiness’ (see Section 2.5). The null hypothesis that giant clams do not aggregate, but rather exhibit haphazard and undirected movement, was tested by comparing the distribution obtained in the above experiment, the live clam run, to a random walk simulation (Uryu et al., 1996). The simulation was created by designing a model in which individual clams move about randomly and stop when each makes physical contact with at least one other clam. The ceasing of movement upon conspecific contact was justified as a preliminary observation of 28 clumps, consisting of between two and seven clams (total 97 clams) revealed no movement in all but one clam in seven days (see also Senawong, 1970; Paine, 1974; Tan, 1975; Okamura, 1986). A random distribution was also generated, using the random number function in Microsoft Excel 2003, for comparison. A single-factor analysis of variance (ANOVA) was applied to reveal differences between the ‘clumpiness’ index derived for the live clam run and the two simulations (random walk and random distribution), while the Student-Newman-Keuls (SNK) test was used to examine multiple pair-wise comparisons among treatments. A field experiment was conducted within 80 × 80 cm2 enclosures on the reef flat of St John's Island using SCUBA to examine aggregative behaviour of clams in their natural environment. Similar to the aquarium experiment, 36 clams (shell length 25–63 mm) were placed 10 cm apart in a 60 × 60 cm2 square area (n = 4; total 144 clams). The coordinates of clams after 24 h were recorded and the ‘clumpiness’ index obtained. Using a single-factor ANOVA, a comparison was performed between index values derived from the live clam run with the random walk and random distribution simulations. Substrate irregularities may have been a modifying factor in structuring movement patterns and the final distribution of clams (Morton, 1962; Uryu et al., 1996; Legendre et al., 1997). To test this effect, we generated topographical profiles of the substratum using depth measurements of two experimental grids to obtain 10 × 10 data points in each square area. Correlation coefficients were obtained between clam occurrence in each cell and the substrate profiles to detect possible association between topography and clam positions.

2.4. Attraction and chemotaxis among giant clams To test for attraction of juvenile clams to conspecifics, their movement was recorded with respect to a fixed ‘target’ in the circular tanks on roughened acrylic sheets. The target object was kept in position by a 3 mmhigh PVC ring attached to the acrylic sheet with an epoxy sealant, while the test clam was positioned in the middle of the circular acrylic sheet 10 cm away (n = 30). Lines were drawn on the acrylic platform to mark the initial position of the test clam. Four objects, plus one control treatment, were used as targets, i.e. (i) live clam; (ii) fouled clam shell; (iii) foul-free clam shell; (iv) random inanimate object such as a rock of comparable size; and (v) none (empty ring). All clams used in each sample (shell length 23–62 mm) had pair-wise differences of b 2 mm in shell length. After 24 h, clam movements with reference to targets (towards, away from, or neutral) were recorded and the resultant 3 × 5 frequency table (‘towards’, ‘neutral’ and ‘away’ vs ‘live clam’, ‘fouled clam shell’, ‘foul-free shell’, ‘inanimate object’ and ‘none’) was analysed with the Pearson Chisquare test. Distance moved from the origin of each clam was also measured to determine whether different targets and clam size affect movement. Data were compared among treatments using a single-factor analysis of covariance (ANCOVA; 5 levels), adjusting for clam length as covariate, followed by post-hoc multiple comparisons performed using the StudentNewman-Keuls test. A choice experiment using a bidirectional water inflow system was designed and fabricated to determine possible chemotaxis of clams in the presence of conspecifics (Morton, 1962). Two 1-l source containers were placed at both ends of a Teflon-coated metal tray (length 28 cm × width 9 cm × height 6 cm). Rubber hoses regulated by taps supplied seawater from the source containers into the tray at two opposite directions (Fig. 2); rate of water inflow was set at 0.5 l min− 1 . The entire setup was placed in an aquarium tank that constantly provided the source containers with fresh filtered seawater and drained the overflow. Eight clams were placed in a randomly selected container, while a test clam was positioned in the middle of the experimental tray (n = 30). A control treatment without any clams placed in either source container was also set up. Clams tested were between 28 and 63 mm in shell length; lengths of all clams in each replicate differed by b 1 mm. Movement of each test clam in relation to the clam effluent source was observed after 3 h and analysed with the Pearson Chi-square test (3 × 2 frequency table;

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Fig. 2. Diagram of chemotaxis experiment, displaying treatment in which clams were placed in one of two source containers.

‘towards’, ‘neutral’ and ‘away’ vs ‘clam effluent’ and ‘control’).

3. Results 3.1. Clam movement and phototaxis

2.5. Data analyses Statistical analyses, including Pearson Chi-square test, t-test, ANOVA, ANCOVA and SNK test, were performed using the software STATISTICA 6.0 (StatSoft); data were transformed for homogeneity of variances, evaluated using Cochran's test (Winer, 1971), if necessary. Spatial pattern analyses were carried out with the software FragStats 3.3 (McGarigal et al., 2002) that provided an index for ‘clumpiness’, i.e. CLUMPY. The index is computed as the deviation of proportion of clams from one that is expected for a random distribution, and is given by 8 Gi −Pi > < i CLUMPY ¼ G P−P > : i i 1−Pi 2

if Gi bPi b0:5 otherwise

Movement was observed in clams ranging from 10 to 313 mm in shell length, with displacement of ∼ 20 cm in three days recorded for the largest individual. Clams with lengths of 10 to 22 mm that were attached with byssus were able to climb up the sides of aquarium tanks. Timelapse images taken of clams from the ventral perspective, aided by observations from dissections, showed the foot protruding out of the shell through the byssal orifice to contact the substrate for movement. Quick contraction of clam valves also generated a force that created horizontal movement. Chi-square test results reveal a highly significant association between type of movement and presence of attachment (χ22 = 216.93; p b 0.001). All unattached individuals moved, with all but one exhibiting translation; 30.8% of byssally attached clams also moved, with more exhibiting rotation (26.6%) than translation (4.2%) (Fig. 3).

3

6 7 gii  7 where Gi ¼ 6 4 P 5 m gik −ei k¼1

and Pi is the proportion of the grid occupied by type i individuals, gii the number of aggregations of type i individuals, gik the number of adjacencies between individuals of types i and k, and ei is the minimum perimeter of type i individuals when they are maximally aggregated (McGarigal et al., 2002). It has a range that encompasses the following distribution patterns: (i) maximally disaggregated: CLUMPY = − 1; (ii) randomly distributed: CLUMPY = 0; and (iii) maximally aggregated: CLUMPY = 1.

Fig. 3. Bar charts showing number of clams exhibiting translation, rotation and remaining stationary in each treatment (n = 120).

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3.2. Aggregation in giant clams

Fig. 4. Bar charts showing number of clams moving with respect to direction of light source (n = 40).

No association between light treatment and direction of movement was identified by Chi-square analysis (χ42 = 0.56; p N 0.05). Clams did not preferentially migrate toward the single light source in the first treatment; both control treatments (light from two opposite directions, and no light) had comparable numbers moving in both directions (Fig. 4). As the direction of light source did not affect locomotive behaviour, there was no necessity for subsequent experiments to account for light influence.

For both aquarium and field aggregation experiments, there were significant differences in mean ‘clumpiness’ index among the three procedures performed, i.e. live clam run, random walk and random distribution (ANOVA, aquarium: p b 0.01, field: p b 0.001) (Table 1; Fig. 5). SNK comparisons showed that clams were more aggregated in the aquarium live clam run (mean CLUMPY = 0.1202 ± S.E. 0.0245) than both random walk (0.0372 ± 0.0157, p b 0.01) and random distribution simulations (0.0195 ± 0.0132, p b 0.01), with no significant difference between the simulations. In the field study, mean CLUMPY was higher in the live clam run (0.2087± 0.0137) than the simulations (p b 0.001). Random walk (−0.1120± 0.0229) resulted in less clumped distribution than random distribution (0.0276 ± 0.0111, p b 0.001). The field grids also had higher mean CLUMPY results compared to the tank grids. Substrate profiles of two field grids are illustrated in Fig. 6, with respective clam occurrences represented by sharp peaks on the surface plots above them. No correlation was found between the

Table 1 Single-factor analyses of variance and post-hoc Student-NewmanKeuls tests comparing mean ‘clumpiness index’ (CLUMPY) among three clam locomotion procedures performed for aquarium (a) and field (b) experiments Source of variation

SS

d.f. MS

F

p

(a) Aquarium experiment Procedure 0.0442 2 0.0221 10.8193 0.0012 Residual 0.0306 15 0.0020 Cochran's test: C = 0.5899; p = 0.3712 SNK test: S.E. = 0.0183; ‘Live clam run’ N ‘Random walk’ (p = 0.0064) ‘Live clam run’ N ‘Random distribution’ (p = 0.0012) ‘Random walk’ = ‘Random distribution’ (p = 0.1973) (b) Field experiment Procedure 0.2068 2 0.1034 93.0160 b0.0001 Residual 0.0100 9 0.0011 Cochran's test: C = 0.6287; p = 0.4687 SNK test: S.E. = 0.0166; ‘Live clam run’ N ‘Random walk’ (p = 0.0002) ‘Live clam run’ N ‘Random distribution’ (p = 0.0002) ‘Random distribution’ N ‘Random walk’ (p = 0.0004)

Fig. 5. Bar charts showing: (a) mean ‘clumpiness’ index (CLUMPY) for procedures performed on clams placed on square grid in the aquarium (n = 6); (b) mean CLUMPY for field procedures performed on clams placed on square grid (n = 4). Error bars indicate standard error.

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Fig. 6. Three-dimensional charts showing clam distribution and substrate profile of two replicate field grids, A and B. Spikes in upper chart represent clam positions.

positions of clams and substrate topography (grid A: r = 0.04; grid B: r = 0.05). The clams were not situated preferentially on any particular terrain features, such as lower areas and depressions.

3.3. Attraction and chemotaxis among giant clams In the attraction tests using fixed targets, Pearson Chisquare analysis revealed a marginally significant association between treatment and direction of movement (χ82 = 15.17; p = 0.0559). The proportion of clams moving toward the live clam target (80.0%) was greater than Table 2 Single-factor analysis of covariance of mean distance moved by clams among treatments, adjusted for clam length as a covariate. Table includes test for homogeneity of slopes of clam length and post-hoc Student-Newman-Keuls tests among treatments

Fig. 7. Bar chart showing: (a) number of clams moving with respect to various ‘targets’ (n =30); (b) mean distance of clam movement among treatments with different targets (n= 30). Error bars indicate standard error.

Source of variation

SS

Homogeneity of slopes Target Clam length Target × clam length Residual

d.f. MS

F

p

1788 4 447 0.7887 0.5344 77,935 1 77,935 137.5362 b0.0001 1313 4 328 0.5792 0.6782 779,332 140 567

ANCOVA adjusted for clam length Target 9060 4 2265 4.0445 0.0039 Clam length 78,215 1 78,215 139.6613 b0.0001 Residual 80,645 144 560 Regression analysis: R2 = 0.5278; adjusted R2 = 0.4975 Cochran's test: C = 0.2432; p = 0.6485 SNK test: S.E. = 4.3205; “Live clam” N “Fouled shell” ( p = 0.0064) “Live clam” N “Foul-free shell” ( p = 0.0049) “Live clam” N “Inanimate object” (p = 0.0019) “Live clam” N “None” ( p = 0.0094) All other pairwise comparisons not significant ( p N 0.9)

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Fig. 8. Bar chart showing number of clams moving with respect to direction of clam effluent (n = 30).

movement away from it (10.0%), and this occurred more often than with other target objects (Fig. 7a). Among treatments with non-living targets, distribution of clam movements was similar. ANCOVA described significant difference in the final distance between the test clam and its initial position, adjusted for clam length as covariate (p b 0.01) (Table 2; Fig. 7b). The SNK procedure revealed a greater amount of movement when the target was a live clam, as compared to all other targets (p b 0.01), which did not differ significantly among one another (p N 0.05). Slopes of distance moved by clam against the covariate were homogeneous among treatments (p N 0.05); regression analysis detected a negative correlation between mobility and clam length (R2 = 0.528). For the chemotaxis choice experiment, Pearson Chisquare test results reveal a significant association between source of clam effluent and direction of movement (χ22 = 15.24; p b 0.01). Only one clam (3.3%) moved away from the effluent of conspecifics, with the majority (80.0%) moving toward water emerging from the source container containing clams (Fig. 8). The control treatment, however, had similar numbers moving in both directions (towards: 33.3%; away: 36.7%; center: 30.0%). 4. Discussion This study confirms that movement and aggregation in T. squamosa do occur. Our observations expand the recorded range of giant clam sizes known to exhibit horizontal movement, i.e. juveniles of 10 to 20 mm clam length in T. crocea (Yonge, 1936), to adults up to a length of 313 mm. The two means of movement observed incorporated typical bivalve motion along the

substratum (foot action) and swimming-type behaviour (valve contraction). This combination could be a consequence of reduction in foot size relative to the enlarging shell, as well as loss of the anterior adductor muscle and anterior pedal retractor in the course of Tridacnidae evolution (Yonge, 1936, 1962). The use of valve adduction for movement supplements foot action in the tridacnid ancestor that diverged from present-day Cardiidae (Stasek, 1962; Ansell, 1967, 1969; Maruyama et al., 1998; Campbell, 2000). The range of 10 to 20 mm shell length reported for vertical climbing in T. crocea (Yonge, 1936) is similar to that found in T. squamosa, i.e. 10 to 22 mm. Locomotive behaviour in bivalves enables them to locate ecological environments that could enhance their survival (Tan, 1975; Waller et al., 1999). The ability of an individual to move in search of suitable substratum and hydrodynamic conditions determines its fitness; this is especially so for populations exposed to physical disturbances caused by natural events or anthropogenic activities (Amyot and Downing, 1997; Waller et al., 1999). Movement was reduced but not eliminated by byssal attachment to the substratum and was probably a result of previously-laid byssus being self-removed, as observed in the mussel Hormomya mutabilis (Senawong, 1970). This mechanism may be more important in translation, as rotation only requires the peripheral elastic byssal threads to be stretched as the clam changes orientation about the central attachments (Davenport and Wilson, 1995; Bell and Gosline, 1996). This would explain why rotation, rather than translation, was more frequently observed in attached clams. Despite possibilities of thread removal and stretching of byssal material most clams did not move, suggesting higher cost involved in movement when attached than when unattached (Davenport and Wilson, 1995). There was no significant association between direction of light source and clam movement pattern, contrary to the algal symbiont's phototactic tendency (Hollingsworth et al., 2005). Several hermatypic coral species (the other major zooxanthellate taxa) have altered behaviours as a result of the symbiont's positive tactic movements. This integration of both host and symbiont responses suggest a coadaptation to light exposure for an overall mutual increase in fitness (e.g. Vereschi and Fricke, 1986; Levy et al., 2006). Our results indicate that this does not occur between T. squamosa (shell length 32–52 mm) and their photosynthetic algae. The cost of movement of the entire shelled animal may be too high for a possible increased rate of photosynthesis to compensate. Juvenile clams clearly aggregated in both the tank and field experiments. The index scores were higher for living

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clams than haphazard ‘clam’ occurrences or random walk simulations, corroborating casual observations made by McMichael (1974) and Yamaguchi (1977). Clumping of clams in wild populations has never been recorded in real time, but the results of the field experiment suggest that this is a natural behaviour. Clumping appears to be more pronounced in the field than in tanks, implying that the addition of a multitude of biotic and abiotic factors in the field can lead to a net change in the degree of clam aggregation. Potential environmental influences include substrate heterogeneity (Legendre et al., 1997), benthic currents (Cosson et al., 1997) and proximity to potential predators (Côté and Jelnikar, 1999). Our results, however, show no correlation between clam positions and substrate topography in the field grids, and that clams did not prefer any distinguishable terrain features. The increase in clam aggregation is therefore not a result of topographic variability. The ‘clumpiness’ index obtained for the random walk simulation was significantly lower than the live clam run. In the field aggregation experiment, random walk resulted in a distribution that was less clumped than random because the time interval given for the test (24 h) was insufficient for ‘clams’ to move out of the regular formation in which they were initially positioned. Clearly, the simulations did not reflect the true pattern of movement by each clam. This contrasts with the behaviour of the freshwater mussel Limnoperna fortunei, for which a similar comparison of real patterns and simulations was carried out, but resulted in more intense aggregations during the simulations (Uryu et al., 1996). The lower-than-expected index obtained by random walk presents a strong argument for the contribution of intraspecific attraction between giant clams in generating aggregations. The hypothesis that clams move toward conspecifics was supported by a non-significant association between target treatment and direction of movement in the attraction experiment. However, the number of clams moving toward the live clam was eight times that of numbers moving away and staying in the centre, as opposed to the approximately similar distribution of movements in other treatments. There was a negative correlation between distance moved by a giant clam and its clam length; smaller individuals departed further from their original positions in 24 h than larger ones. There have been some studies showing the greater movement ability in younger, smaller bivalves (Senawong, 1970; Uryu et al., 1996; Legendre et al., 1997), but as all clams in this study were obtained from a single spawning event, it was, rather than age, that affected mobility. The reduced locomotion in larger individuals is most likely a result of the

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decrease of foot size to shell-weight ratio as clam size increases (Yonge, 1936; Uryu et al., 1996; Waller et al., 1999). It has been shown previously that green mussels, Perna viridis, have a greater tendency to move when they are not in close proximity to conspecifics (Tan, 1975). This behaviour may be important in the formation of clumps, as a mussel ‘explores’ its surroundings in search of a more suitable location (Seed and Richardson, 1999) — possibly in the direction of another mussel with which to aggregate. In fluted giant clams, however, data from the target attraction experiment showed that there was more movement in the test clam when the target was a live conspecific, compared to other treatments. Unless there is another clam in the vicinity, T. squamosa does not appear to ‘explore’ the surroundings more by intensifying movement. Rather, locomotion may be increased if the benefit of clumping exceeds the energetic cost associated with increased clam movement. This is likely to occur when at least one clam is detected in close proximity. The clams in the present study showed a preference for conspecific effluent by moving toward its source in the bidirectional water inflow experiment. Since the effluent from source containers was the only way that any odour can reach the test clam, the attractant had to be in the form of water-borne chemical compound(s) carried from the eight clams into the experimental tray (Morton, 1962). To our knowledge, this is the first direct test of positive chemotaxis between conspecifics in the Bivalvia, providing support for non-random attractive movement as the primary proximate mechanism of aggregation in giant clams. This may account for the higher degree of aggregation in field grids than in the tanks as bottom currents could assist in transporting clam odour among the test subjects on the reef (Weissburg and Zimmer-Faust, 1993). In a large tank of water with minimal fluid movement, concentrations of attractant received by each clam from its immediate neighbours are approximately equal, resulting in a reduced ability to determine their locations. The diffusion and accumulation of odours emitted by all clams can also give rise to a roughly uniform concentration of attractants in the water column (Weissburg, 2000), rendering each animal less able to locate higher clam densities. Furthermore, when fluid motion is established in the tank due to temperature and salinity variations, they tend to be unpredictable and multidirectional (Turner and Stommel, 1964). Aggregation as a result of clam movement is a tradeoff between the supposed costs and benefits of being in close proximity to conspecifics. Costs may include enhanced risk of detection by predators, increased likelihood of parasite dispersal, and intraspecific

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competition (Krause and Ruxton, 2002). But clump formation could also serve several fitness-conferring ecological functions, such as defence against predation, physical stabilisation, and increased reproductive success (Jackson, 1977; Shanks and Wright, 1986; Parrish and Edelstein-Keshet, 1999). Aggregation as an antipredatory defence strategy has been well-studied in bivalves (e.g. Lin, 1991; Reimer and Tedengren, 1997). As predators have upper limits to the rate at which they can catch and consume prey, being in clumps reduces the risk each animal faces as compared to life as a solitary individual (Krause and Ruxton, 2002). Experimentally, crab predation rates have been shown to decrease at high clump densities of the ribbed mussel Geukensia demissa (Bertness and Grosholz, 1985), while juvenile bivalves could be protected among adult shells that offer substantial spatial refuge (Kitching et al., 1959). Aggregation can also be induced by the threat of predators, for example, blue mussels, Mytilus edulis, form clumps faster in seawater that has been conditioned with a lobster predator compared to control water (Côté and Jelnikar, 1999). The other well-supported function of benthic animals aggregating is physical stabilisation against abiotic stresses (e.g. Harger, 1970; Harger and Landenberger, 1971). Common problems associated with water and ice movements faced by animals can be reduced as a result of aggregative behaviour. Bivalve individuals within clumps receive physical support from neighbouring conspecifics, and there is generally a reduction in animal surface area exposed to mechanical action (Seed, 1969). These factors guard them from winter ice dislodgement (Kitching et al., 1959) and displacement by wave forces (Harger, 1970). In species that produce byssus, individuals may obtain firmer attachments to the substratum by merging byssal threads with those of adjacent conspecifics (Bertness and Grosholz, 1985). Furthermore, the risk of desiccation can be reduced as byssal threads trap moisture (Seed, 1969); this is important for intertidal bivalves that may be exposed during very low water tides, including T. squamosa. Aggregations are therefore better able to survive in high energy and/or desiccationprone environments than solitary individuals. Reproductive success can be enhanced by aggregative behaviour in broadcast spawning organisms that externally fertilise (Yamaguchi, 1977; Levitan, 2002). One primary constraint on the success of spawning is dilution of gametes (Metaxas et al., 2002), hence it is of no surprise that there is evidence showing positive correlation between fertilisation success and densities of spawning individuals among various aquatic taxa (Pennington, 1985; Downing et al., 1993; Levitan,

2002). Tridacnid clams are primarily seasonal synchronous spawners within populations (Heslinga et al., 1984; Shelley and Southgate, 1988), although some have variable times of gamete maturation extending up to four months (Tan and Yasin, 2000). After spawning, sperm may remain active for a maximum of an hour, but fertilisation usually occurs within 15 min of egg release (Braley, 1992; Ellis, 1999). The synchronicity of spawning and the short period available for fertilisation suggest that clustering behaviour could increase the reproductive success of clams. Theoretically, potential reproductive benefits of aggregating are significant in giant clams. Results from the present study also reveal that giant clam mobility decreases with increasing shell size. Juvenile clams, by moving toward one another while still exhibiting high levels of locomotion, can form aggregations before movement becomes limited with growth. Worldwide, natural clam populations are declining due to decades of over-exploitation fueled by the notorious aquarium, ornamental and seafood trades in several East Asian states (Wells et al., 1983; Lucas, 1994; Heslinga, 1996; Ellis, 2000; Mingoa-Licuanan and Gomez, 2002). As the situation exacerbates, there is a reduced opportunity for conspecific clumping to occur since the clams become extremely dispersed. In Singapore, for instance, a survey of seven coral reefs with a total area of almost 10,000 m2 uncovered only 15 individuals of T. squamosa (Guest et al., in review). Unfortunately, if aggregation enhances reproduction, local stocks could be facing increased vulnerability to Allee affects, resulting in local extinction events sooner than would be expected for non-aggregating animals (Reed and Dobson, 1993; Courchamp et al., 1999; Stephens and Sutherland, 1999; Knowlton, 2001). Conservation of giant clams often involves restocking coral reefs (Lucas, 1994; de Vicose and Chou, 1999; Mingoa-Licuanan and Gomez, 2002). To increase the chance of transplanted clams surviving in sparselypopulated reef sites, potential reproductive benefits of aggregations need to be taken into account and used to the advantage of resource managers (Lucas, 1994; Sutherland, 1998). Currently, most reseeding operations have emphasised the transplanting of clams to a large number of sites (Mingoa-Licuanan and Gomez, 2002). However, it may be beneficial to repopulate fewer sites with more clams each (Lucas, 1994). With a high density of clams, a refuge may be created as a source of larvae and recruits for natural repopulation of neighbouring reefs (Green and Craig, 1999; Gilbert et al., 2005). Prior to transplantation, the grow-out stage of giant clam farming has an important influence on the survival and

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number of clams that may finally be used in the reseeding operation (Braley, 1992). Current mariculture methods advise that clumped clams should be redistributed (Ellis, 2000). This is based on the fact that under physical pressure of neighbouring clams, valves can become distorted and deformed, with overall shell growth restrained (Harger, 1972; Bertness and Grosholz, 1985; Ellis, 2000). We propose an alternative viewpoint, that such a procedure be balanced with the organism's natural aggregative response that might play an ecologically important role. The spatial condition of a bivalve greatly affects its survival, and mobile giant clams will move to seek out more favourable locations (Burks et al., 2002). By constantly separating the clumps, individual clams repeatedly move and aggregate, expending energy that could be used for growth and respiration. This may result in undesirable stunted growth and undue stress in cultured clams. In conclusion, chemical signalling occurs among juvenile T. squamosa that increases the horizontal movement of individuals toward higher concentration of an unidentified clam odour. They eventually contact adjacent conspecifics and cease movement, giving rise to clam aggregations. Such behaviour suggests that there is some benefit derived by living in groups. The risks of predator detection, parasite load and intraspecific competition have potentially been surpassed by benefits such as defence against predation, physical stabilisation and enhanced reproductive success due to proximity of conspecific neighbours. Acknowledgements TMSI kindly supplied the study organisms and allowed us to use their aquarium and laboratory facilities. We thank L.M. Chou and members of the Marine Biology Laboratory, National University of Singapore, for their help and support. Assistance in the field by A. Aliyar, M.W.P. Chng and C.S. Lee are greatly appreciated. Thanks also to D. Li, R. Meier, K.P Reddy and K.S. Tan for ideas and advice. [RH] References Ackerman, J.D., Sim, B., Nichols, A.J., Claudi, R., 1994. A review of the early history of zebra mussels (Dreissena polymorpha): comparisons with marine bivalves. Can. J. Zool. 72, 1169–1179. Akberali, H.B., Davenport, J., 1982. The detection of salinity changes by the marine bivalve molluscs Scrobicularia plana (da Costa) and Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 58, 59–71. Alcala, A.C., 1986. Distribution and abundance of giant clams (family Tridacnidae) in the South-Central Philippines. Silliman J. 33 (1–4), 1–9. Allee, W.C., 1931. Animal Aggregations. A Study in General Sociology. University of Chicago Press, Chicago.

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