Sand elimination by the Asiatic hard clam Meretrix meretrix (L.): influences of temperature, salinity and season.

ABSTRACT Clams, being filter-feeders that burrow into sand, tend to accumulate sand in their body tissues. Depuration reduces the clams' gut contents and eliminates sand from different body fractions, providing some cleansing and ensuring that they are safe for human consumption. The Asiatic hard clam Meretrix meretrix (Bivalvia: Veneridae) is a commercially important species inhabiting sand flats of Hong Kong and Southeast Asia. In this laboratory study, the effects of temperature and salinity on the rate and profile of sand elimination in M. meretrix were studied during winter and summer using a 2 x 2 factorial design (i.e., two temperatures 20[degrees]C and 30[degrees]C, and two salinities 15%and 30%). Sand removal rate was measured in terms of a decrease in inorganic content (i.e., ash) over a 72-h period. In general, sand was removed from the gut and body tissues and transferred to the external body fluid for subsequent discharge to the ambient water. Meretrix meretrix exhibited significantly lower sand removal rates in summer than in winter across all treatments, whereas the highest sand removal rate was observed at 20[degrees]C and 15% in both seasons. The sand removal rate of M. meretrix is, therefore, not only affected by temperature and salinity, but also strongly associated with natural, seasonal, effects. These findings have important implications for optimizing conditions for sand elimination of this species for the shellfish industry in Asia.

KEY WORDS: depuration, salinity, sand elimination, season, temperature

INTRODUCTION

Sandy shore bivalves burrow into sand as an adaptation to protect themselves from wave impact, temperature fluctuations, desiccation, and predation (Nybakken 1997). As filter feeders, they feed during high tide using their siphons, which extend to the sand surface to collect microscopic organisms and particulates from the water column (Morton 1979). Ingested particles, containing both organic and inorganic matter, are either digested in the gut or expelled as faecal pellets (Hylleberg & Gallucci 1975, Ruppert & Barnes 1994). In addition sand particles, together with other unwanted particles such as viruses, bacteria, and pollutants, will be trapped in different organs such as the gills, gut, and adductor muscles (Richards 1988, Savari et al. 1991, Sobsey & Jaykus 1991, Somerset 1991, Ho & Kim 1995, Ariza et al. 1999). When the clams are sold for human consumption, the tissues that contain sand can be unpleasant to eat and also pose a health risk. As a common practice the clams are, therefore, allowed to clean themselves in clean seawater (i.e., depuration) to remove the sand in their body (Richards 1988, Savari et al. 1991).

In bivalves, significant effects of salinity have been reported on mortality (Mane 1978, Morton & Chan 1990), growth (Robert et al. 1988, Hutchinson & Hawkins 1992), and cardiac and siphonal pumping activity (Akberali & Davenport 1981). Salinity is especially important in controlling the effectiveness of depuration by sandy shore bivalves. Richards (1988), for example, demonstrated that salinities between 22% and 31% were optimal for microbial deputation by the hard clam Mercenaria mercenaria (L.), soft-shell clam Mya arenaria (L.), and hen clam Venus gallina (L.).

Apart from salinity, the depuration rate of bivalves is also influenced by water temperature, suggesting that it may be associated with temperature-dependent metabolic activities. Previous studies have shown that growth (His et al. 1989), respiratory responses (Hicks & McMahon 2002), and filtration rate (Walne 1972) of bivalves are temperature-dependent. Richards (1988) found that the depuration of viruses by the hard clam Mercenaria mercenaria (L.) was improved by increasing temperature, and a similar result was observed in a study on the elimination of coliphages by the mussel Mytilus edulis (L.) (Power & Collins 1990). Additionally, Richards (1988) investigated the seasonal variation of microbial depuration by temperate clams in terms of the combined effect of salinity and temperature and revealed that viruses were more effectively depurated from the clams during the summer and fail months than during the winter.

Although the depuration of pathogens (e.g., viruses; Carter & Cantelmo 1990, Ho & Tam 2000), soluble pollutants (e.g., hydrocarbons and heavy metals; Denton & Jones 1981, Jovanovich & Marion 1987, Savari el. al. 1991, Reinfelder et al. 1997, Ariza et al. 1999), and phytotoxins (Blanco et al. 2002) in bivalves has widely been studied, information on the elimination rate of insoluble, sand-like particles in clams and cockles is scarce. Indeed, there are only a few reports on sand elimination rates in mussel, clam, and cockle species in relation to their storage and handling in the shellfish industry (De Vooys 1987, Richards 1988, NSSP 1990. Somerset 1991).

The current study was therefore designed to investigate the rate of sand elimination by the commercially important, Asiatic hard clam Meretrix meretrix (L.) in clean seawater at four different temperature/salinity regimes during winter and summer, respectively. The results from such a study are important for optimizing conditions for sand elimination of this species for the shellfish industry in Asia.

MATERIALS AND METHODS

Sampling and Experimental Design

Meretrix meretrix is one of the largest clam species among the Venus-shells (Veneridae) and very common on sheltered sand flats in Hong Kong, South China, India, Vietnam, Thailand, and the Saudi Arabian Gulf (Morton & Morton 1983, Jayabal & Kalyani 1986, Nhan et al. 1999, Vazquez el al. 1991). As a commercially important clam species in Asia, it has widely been cultured in China and Thailand (Ho & Zheng 1994, Ho & Kim 1995).

In the current study, adult M. meretrix were randomly collected from Shui Hau in Lantau Island, Hong Kong (Grid reference: 22[degrees]13'N, 113[degrees]55'E) during low tides in winter (24 February 2003; 75 individuals; shell length: 50.01 [+ or -] 4.13 mm, mean [+ or -] SD) and in summer (5 June 2003; 75 individuals; shell length: 54.9 [+ or -] 7.3 mm). Clams were collected using a specially constructed clam-rack commonly used by fishermen in Hong Kong and South China (Morton & Morton 1983).

Hong Kong has a strongly seasonal, monsoonal climate with a hot, wet summer and cool, dry winter (Kaehler & Williams 1996). Seawater temperature and salinity were measured using a thermometer and a refractrometer (ATGAO, S/Mill-E, Tokyo, Japan). The seawater temperature and salinity in the field were 15.0[degrees]C and 32.0 [per thousand], in winter and 29.0[degrees]C and 14.0 [per thousand] in summer. Such a considerable difference in salinity between the two seasons was mainly due to the close proximity of the site to the Pearl River estuary, which receives a huge amount of freshwater input during summer (Morton & Wu 1975). The collected clams were transferred to the laboratory within 2 h of collection. On arrival, the clams were divided into four groups and held in 1 L, aerated glass tanks filled with 500 mL of artificial seawater (Kent Marine Salt, Kent Marine Inc., Acworth, Georgia) at the same salinity level as measured at the collection site and at room temperature (20[degrees]C). The experimental ranges of temperatures and salinities were based on the natural ranges in the environment (Morton & Wu 1975, Morton & Morton 1983). The clams were acclimatized to four combinations of two temperatures (20 [+ or -] 0.2[degrees]C and 30 [+ or -] 0.4[degrees]C, mean [+ or -] SD) and two salinities (15 [+ or -] 0.8 [per thousand] and 30 [+ or -] 0.4 [per thousand]). The water temperature of the 30[degrees]C treatments was adjusted by increasing 4[degrees]C every 3 h using a water bath with a temperature controller, whereas the 20[degrees]C treatments were maintained at room temperature. Salinity was adjusted by decreasing or increasing values by 5 [per thousand] every 3 h until the desired levels were reached. This was achieved by renewing the seawater with artificial sea salts. A control group of 15 individuals was randomly selected at the beginning of the experiment to determine the initial inorganic content of the clams prior to depuration.

For each treatment group, there were three 1-L glass tanks, each holding 5 animals ([SIGMA]n = 4 treatments x 3 tanks x 5 clams = 60 clams). Water salinity and temperature were kept constant for each treatment and monitored twice daily. Experimental water was renewed daily. No food was provided and a 12-h light: 12-h dark cycle was maintained throughout the experiment. In a preliminary study, Lui (2003, unpublished data) observed that M. meretrix individuals removed >65% of sand from their body after 72 h in artificial seawater at 20[degrees]C and 32 [per thousand] (sand remaining [%] = 202.2 x [exp.sup.-4.5 x +] 34.2, where t is the duration of sand removal in hours). The clams were, therefore, allowed to depurate over 72 h in the current study, after which all animals were collected for dissection, condition index, and inorganic content measurements.

Dissection of Clams

To study the sand elimination profile and pathway, sand content in three different body compartments, namely the external body fluid, the gut, and the remaining soft body tissues, were determined. The animals in their shells were thoroughly scrubbed with a stiff brush in running tap water, rinsed with distilled water, and left to dry on clean paper towels. Shucking was achieved by inserting a dissecting knife at the site of the adductor muscle. After the shell valves were opened, any liquid was drained and collected in a preweighed aluminum container. This sample was designated as the external content (EC). The digestive tract was then carefully dissected and designated as the gut content (GC) and placed in a preweighed aluminum container. The remaining body tissues were also placed in another preweighed aluminum container and designated as the body content (BC).

Inorganic Content Analysis and Condition Index

Five empty, preweighed aluminum containers were used as controls for the weighing procedure. All samples, along with the empty shells, were dried in an oven at 80[degrees]C for at least 48 h until a constant dry weight was achieved. For each animal, dry weights of all the components (i.e., EC, GC, and BC) and empty shells were weighed using an electronic balance ([+ or -] 0.00001 g; OHAUS Analytical Plus, Nanikou, Switzerland). Dried samples were then ignited at 500[degrees]C in a muffle furnace for 4 h (Takada 1995) and the remaining ash content weighed. Three readings were taken for each sample to ensure a precise mean value for representing the dry ash weight. The inorganic content of each component was calculated as the percentage of the ash weight of the total dry weight of the whole soft body tissues. Total inorganic content percentage (TIC, %) of each clam was calculated using the following equation:

(1) TIC (%) = (Total Ash Weight/Total Dry Soft-Body Tissue Weight) x 100%

where total ash weight was calculated by summation of the ash weights from all of the three fractions (EC, GC, and BC) and total dry tissue weight was the sum of the dry weights of the gut and body tissues.

The condition index (CI) was calculated using the following equation described in Beninger and Lucas (1984) and expressed as g tissue per g shell weight:

CI = Dry Soft-Body Tissue Weight/Dry Shell Weight (2)

The Rate of Sand Elimination

The rate of sand elimination (mg [g.sup.-1] [h.sup.-1] for each species was calculated using the following equation:

(3) Rate = (TI[C.sub.final] - TI[C.sub.initial]/Depuration Time

where TI[C.sub.final] is the total inorganic concentration (mg [g.sup.-1] dry soft-body weight) at the end of the experiment over the total depuration time, and TI[C.sub.initial] is the initial total inorganic concentration (mg [g.sup.-1] dry soft-body weight) at time zero. The results were also expressed as percent sand remaining, which was a ratio of sand content between the control claims (i.e., initial) and each treatment group after 72 h depuration.

Statistical Analysis

Data were tested for homogeneity of variances using Levene's test (SPSS version 10.0). Two-way analysis of variance (ANOVA) was used to test the effect of the treatment (five levels: control and four temperature/salinity regimes) and season (two levels: winter and summer) on the condition index. To compare the inorganic content in each individual components (i.e., EC, GC, and BC) among the control and the four regimes in each season, one-way ANOVAs were used followed by post hoc Tukey's multiple comparison tests to identify significantly different means. Finally, a three-way ANOVA was used to test the significance of the effects of temperature (two levels), salinity (two levels), and season (two levels) on the sand removal rate.

RESULTS

Condition Index

As no food was provided during the depuration period, the condition index (CI) of M. meretrix decreased significantly after the 72 h depuration for the four treatments in both seasons (Fig. 1; two-way ANOVA: treatment effect, [F.sub.4, 19] = 35.64, P < 0.001). The CI values were significantly lower in summer than in winter across all of the control and treatment groups (Fig. 1; two-way ANOVA: season effect, [F.sub.1, 19] = 34.74, P < 0.001).

[FIGURE 1 OMITTED]

Inorganic Content in Different Body Compartments

Before depuration, the distribution of sand in the control group of M. meretrix followed the order external body fluid (EC) > body tissues (BC) > guts (GC) during the summer (Figs. 2a, 2c, and 2e). In contrast, higher levels of sand grit were found in BC than EC in the control M. meretrix group during the winter (Figs. 2b, 2d, and 2f). After the 72-h depuration period, the inorganic content in the gut decreased across all treatments in both seasons, although this decline in the clams exposed to 30 [per thousand] and 20[degrees]C was not statistically significant (Figs. 2c and 2d). For treatments under high salinity (30 [per thousand]) regardless of their water temperature and season, the amount of sand depurated was small, and the EC accounted for the largest proportion of overall inorganic content (Figs. 2a and 2b). Meretrix meretrix collected in winter and exposed to the two high-salinity regimes showed a significant increase in inorganic content in the EC over the deputation period (Fig. 2b). In terms of total inorganic contents (TIC) per individual clam (Figs. 2g and 2h), the two low-salinity (15 [per thousand]) treatment groups exhibited significant reductions in their sand contents, whereas the two high-salinity (30 [per thousand]) treatment groups did not show any significant decline in TIC. These results indicate that the speed of transportation of sand from the body tissues and gut to the external fluid for discharge was much slower in M. meretrix exposed to high salinities (30 [per thousand]) than for the other treatment groups at low salinity (15 [per thousand]).

[FIGURE 2 OMITTED]

The sand removal rate of M. meretrix was strongly affected by the different temperature/salinity regimes and season (Figs. 3a and 3b). In general, significantly higher sand removal rates (or lower sand remaining %) were observed at low salinity and low temperature (Fig. 3; Table 1), with the highest elimination rate at 15 [per thousand] and 20[degrees]C for both seasons (Fig. 3). The rate of sand removal was, however, significantly lower in the summer than in the winter (Fig. 3; Table 1). When comparing the magnitude of the differences among the four groups and the F values (Fig. 3b; Table 1), it was clear that salinity was a more important factor affecting sand elimination rates in M. meretrix than temperature and season. This was further supported by the absence of any significant interactions between temperature, salinity, and season on the rate of sand removal (Table 1).

[FIGURE 3 OMITTED]

DISCUSSION

Sand Distribution and Depuration

In this study, sand (as determined by inorganic content) was transported and accumulated in different body compartments. Sand particles either passed through the gut and were discharged as feces or pseudofeces or were transported from the tissues to the external body fluid for subsequent disposal from the edge of the shell. During a 72-h depuration period, a considerable amount of sand particles was defecated from the gut by M. meretrix in both seasons. The inorganic content remaining in the gut might serve several adaptive functions. First, inorganic matter might contain some essential nutrients that are assimilated through the gut or may have microalgal food items bound to their surface (Huz et al. 2002). Second, Chia (1973) demonstrated that sand grains could be used as a weight belt for juvenile sand dollars, Dendraster excentricus (Eschscholtz), that stored these grains in their intestinal diverticulum to help them settle on the sea floor. There is, however, much less information available on the adaptive function of sediment accumulation in burrowing clams.

Salinity Effect

Sand removal rate in M. meretrix was higher at low salinities (15 [per thousand]). A similar result has been reported for the blue mussel, Mytilus edulis, and the hard clam, Mercenaria mercenaria, concerning the depuration of coliphages and Escherichia coli (Richards 1988, Power & Collins 1990). Such an increase in depuralion rate may be attributable to an increase in filtration rate and pupming activity at low salinities. Nonetheless, Richards (1988) also reported that other species, such as the soft-shell clam Mya arenaria (L.), showed a greater depuration rate at higher salinities (25-30 [per thousand]), suggesting that the effect of salinity is species specific. Sobsey and Jaykus (1991) also reported that the range of optimal salinity for effective deputation varies among different bivalve species.

Variation in salinity affects bivalve physiology in a number of ways. Hutchinson and Hawkins (1992) demonstrated that the excretion rate of the oyster Ostrea edulis (L.) increased at low salinities, whereas the effect of salinity on their growth varied, depending on the level of food availability. Shell valve activity in response to variation in salinity may be another factor controlling the rate of sand elimination. Akberali and Davenport (1981), for example, showed that the exhalant siphon and valves of the clam Scrobicularia plana (de Costa) closed at low salinities and thus prevented their pumping activity.

In the current study, M. meretrix removed more sand at low salinities (15 [per thousand]), suggesting that they perform well under lower salinities. Meretrix meretrix occurs on shores in Hong Kong where surface salinities may tall to as little as 2 [per thousand] as a result of the summer monsoons and the influx of freshwater from the Pearl River (Morton & Morton 1983). This ability to survive in low salinities is important for infaunal species, as the salinity of estuarine and intertidal zones can fluctuate on an hourly, daily, weekly, and seasonal basis (Gainey & Greenberg 1977, Leung et al. 2002). This may explain why the effect of salinity on sand elimination in M. meretrix did not vary between winter and summer.

Temperature Effect

Sand elimination by M. meretrix is also temperature-dependent; higher removal rates of sand were generally found at lower temperatures (20[degrees]C). Some shellfish sellers in Hong Kong use lower water temperatures to achieve effective sand depuration by clams, as survival rates of the shellfish are generally higher al lower temperatures such as 17-18[degrees]C (Lui, personal communication). Matthews and McMahon (1999) also demonstrated that the zebra mussel, Dreissena polymorpha (Pallas), and the Asian clam, Corbicula fluminea (Muller), showed a higher tolerance to hypoxia at lower temperatures. In contrast, however, the West African clam, Galatea paradoxa (Born), showed a greater removal of sand at elevated temperatures (Ekanem 2000), whereas the deputation of" E. eoli also increased at higher temperatures in Mytilus edulis (Power & Collins 1990) and the king scallop, Pecten maximus, (L.) (Heath & Pyke 2001), suggesting that such temperature-dependent responses also vary between species.

Generally, in bivalves, higher water temperatures result in increased feeding rates (in terms of filtration rates; Walne 1972) and higher metabolic rates (in aspects of growth; Robert et al. 1988) and oxygen consumption (Hicks & McMahon 2002). Some workers, however, did not find any significant effects of temperature at the organism level (Denton & Jones 1981, De Vooys 1987, Inza et al. 1998, Blanco et al. 2002), and they argue that the deputation process is independent of metabolic rates, as their target species still efficiently depurated over a broad range of temperatures (6.6 20.5[degrees]C). Whether depuration is a temperature-dependent physiologic process in all bivalves species has, therefore, yet to be confirmed.

Combined Effects of Salinity, Temperature, and Season

The effect of temperature on sand elimination by Meretrix meretrix was not as pronounced as the effect of salinity. Meretrix meretrix must tolerate a wide range of salinities on western shores of Hong Kong. The effect of temperature shock (either hot or cold) is, however, likely to be minimal on the shore because they burrow into the sediment where temperatures are more stable as compared with air temperatures (Yang et al. 2003).

In M. meretrir, oplimal sand elimination was achieved at low temperatures (20[degrees]C) and salinitics (15 [per thousand]) in both summer and winter. Although M. meretrix exhibited a higher rate of sand removal in winter than summer, the influence of temperature and salinity was similar between the two seasons. Interestingly, the significantly lower condition indices for M. meretrix in the summer (June) were probably associated with low food (i.e., particulate organic matter) availability in the water column (Wong & Cheung 2003), which may possibly be due to heat stress and the increase in freshwater input from rainfall and surface runoff. In contrast, micro- and macroalgae are more abundant during winter in Hong Kong coastal environments (Morton & Morton 1983, Hodgkiss 1984, Nagarkar & Williams 1999) and may considerably enhance the growth of the clams. In such favorable conditions in winter, the clams not only have better physiologic status to efficiently ingest and assimilate nutrients but are also able to excrete waste and depurate sand more effectively. Jovanovich and Marion (1987) also observed a seasonal variation in the deputation of anthracene by the clam Rangia cuneata (Sowerby); depuration rates remained low from March to August and rapidly increased to the highest levels during the fall spawning period. This seasonality was related to the reproductive cycle of R. cuneata and associated biochemical changes, which in turn affected the physiologic state of the clam.

The oyster Ostrea edulis (L.) shows physiologic variation according to seasonality (Hutchinson & Hawkins 1992). A "winter" physiologic state enables O. edulis to survive at low temperatures and salinites that are normally encountered during the winter months in its shallow coastal water habitat, whereas a reverse physiologic state was recorded in summer. Similarly, siphonal activity of the soft shell clam, Mya arenaria (L.), was higher during May September and then strongly declined from October (Thorin 2000). This variation was mainly attributed to seasonal changes in resource availability, water temperature, or individual reproductive state of M. arenaria. To our knowledge, the natural reproductive cycle of M. meretrix has yet to be established in Hong Kong and South China, although they have been artificially reared or cultured in Southeast Asia. As a result, we are unable to comment on the possible influence of their reproductive cycle on sand elimination.

Conclusions

Sand can be trapped in different body compartments in burrowing bivalves, and this needs to be eliminated for the clam to cleanse itself in the natural environment and for use in the shellfish industry to ensure safety for human consumption. Simple methods for sand elimination using clean seawater or freshwater are currently used by shellfish sellers and seafood restaurant owners worldwide (De Vooys 1987, Carter & Cantelmo 1990, NSSP 1990, Ho & Tam 2000, Heath & Pyke 2001). This pilot study investigated the effect of salinity, temperature, and season on sand elimination in the commercially important clam Meretrix meretrix. The rate of sand removal by M. meretrix was significantly affected by salinity and temperature. The highest rate of sand removal was found at low salinities (15 [per thousand]) and temperatures (20[degrees]C). These optimal depuration conditions were identical in both summer and winter, suggesting that lower temperature and lower salinity were physiologically more favorable regardless of seasonality. Depuration rates in the clams were, however, higher in winter for all treatment groups, and this is probably associated with seasonal variation in the climate, food availability, and reproductive cycle of the clam. Further investigation is, however, required to test whether factors other than salinity and temperature, such as reproductive cycle and food availability, could be important in regulating the rate of sand depuration in M. meretrix.

TABLE 1.
Results of three-way ANOVA on the sand depuration rate
(mg [g.sup.-1] [h.sup.-1]) of Meretrix meretrix exposed
to the four different salinity and temperature regimes
during the summer and winter.

Factor                      df      MS         F        P Value

Temp.                        1   0.174000     8.625    0.010 *
Salinity                     1   4.087000   202.487   <0.0001 ***
Season                       1   0.135000     6.683    0.021 *
Temp. x Salinity             1   0.014140     0.701    0.416
Temp. x Season               1   0.057400     2.844    0.112
Salinity x Season            1   0.001029     0.051    0.824
Temp. x Salinity x Season    1   0.000383     0.019    0.892
Residual                    15   0.020180

Asterisks denote significant factors at * P < 0.05: *** P < 0.001.

ACKNOWLEDGMENTS

The authors thank Cecily Law. Laura Wong, and Lily Ng for their technical support and Cyrus Cheng and Shirley Lui for helping with collecting the samples. Special thanks are also extended to Gray A. Williams, Will Trewhella, Valerie Ho, Justine Tsui, Danny Lau, and Jasmine Ng for their critical reading of earlier drafts of this manuscript.

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KAREN K. Y. LUI (1) AND KENNETH M. Y. LEUNG (1) *

(1) The Swire Institute of Marine Science and Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam, Hong Kong, China

* Corresponding author. E-mail: [e-mail]