Effects of diets, their concentrations and clam size on filtration rate of hard clams (Meretrix lusoria).

ABSTRACT The hard clam (Meretrix lusoria) is one of the most important cultured molluscs in Taiwan. Although it is mainly cultured in ponds and supplemental feeding of artificial feed is commonly practiced, no scientific information on its preferred foods and appropriate feeding strategies are available. Six diets: fishmeal (F), soybean meal (S), commercial hard clam feed (C), microalgae Tetraselmis chui (T) and Nannochloropsis oculata (N) and bread yeast (Y), were fed to hard clams. Each diet was fed at 3 concentrations (20 mg [L.sup.-1], 200 mg [L.sup.-1] and 633 mg [L.sup.-1]) and was fed to clams of 3 sizes (7-11g, 12-17 g and 18-26 g total body weight) to gauge their effects on weight-specific filtration rate and pseudofeces production. Overall, the filtration rates for diets in descending order were S > C > F = Y > N > T. Particle size of the diets played an important, but not the only. role in determining the filtration rate. Diets with regular particle shape (N, T, Y) yield lower filtration rates and higher pseudofeces production than diets with irregular shape (C, F, S). Both filtration rate and pseudofeces production were higher for yeast than for algae (T, N). Filtration rate and pseudofeces production were also higher for the mixed component diet (C) than for single component diets (F, S). Filtration rate, but not pseudofeces production, was higher for the plant-based diet (S) than for fishmeal (F). Both diet concentration and clam size had inverse correlations with weight-specific filtration rate. The effects of all interactions between diets, diet concentrations, and clam sizes on filtration rates were significant, reflecting the complexity of feeding behavior of Meretrix lusoria.

KEY WORDS: clam, Meretrix lusoria, filtration rate, pseudofeces, commercial feed, microalgae, fishmeal, soybean meal

INTRODUCTION

The hard clam (Meretrix lusoria) is one of the most important cultured molluscs in Taiwan. Its reported peak culture area was 7,552 ha in 1994, peak production was 31,517 t in 2003, peak productivity was 4.85 t [ha.sup.-1] in 2002: (Fig. 1). Hard clams were introduced to the northern coast of Taiwan from Japan in 1925 and later extensively ranched in sandy tidal flats, especially on the west coast. Before 1970, hard clams were cultured in ponds (Chen 1984) although it is unknown when this activity first started. In 1980, pond-culture accounted for only 13% of the total culture area. Hard clams were mostly cultured on sandy tidal fiats. After mass artificial propagation was achieved (Chen & Lyuu 1982), total production increased 74% from 9,200 t in 1982 to 16,049 t in 1983 but also the importance of pond-culture. In 2003, around 83% of the culture area was in ponds and 94% of the production was from ponds.

[FIGURE 1 OMITTED]

Various artificial diets, either as supplements or as the main food source for larval, juvenile and adult bivalves have been developed, which include dried algae (Laing et al. 1990, Gladue 1991, Laing & Gil Verdugo 1991, Laing & Millican 1992), preserved algal pastes (Donaldson 1991), microencapsulated diets (Jones et al. 1984, Langdon et al. 1985) and yeast-based diets (Epifanio 1979, Urban & Langdon 1984, Coutteau et al. 1990, 1991). Such feeding, conducted either experimentally or commercially, however, was mostly done in hatcheries or nursery ponds (Southgate et al. 1998), not in growout ponds. In Taiwan, feed accounted for 14% to 37% of total production cost of pond-cultured hard clam (Guo 2003). There are two feed mills in Taiwan producing formulated hard clam feed, which is used by some farmers.

Hard clams have much higher productivity when cultured in ponds than when ranched in sandy tidal flats. In 2003, productivity in the former, 5,480 kg [ha.sup.-1], was 2.2 times higher than that in the latter, 1,710 kg [ha.sup.-1] (Fisheries Administration 2003). In addition, higher stocking densities are used in growout ponds: 1.00 to 1.60 million seed clams (0.8-1.0 g) [ha.sup.-1] versus <1.00 million seed clams [ha.sup.-1], and faster growth occurs in ponds: 6-8 mo versus 1 year to reach marketable size (20 g total body wet weight) (Chen 1984). Organic fertilization with rice bran, chicken droppings, hog manure, and supplemental feeding contribute greatly to the food supply in ponds (Ho 1991). In Taiwan, food organisms used in hatcheries or nursery ponds include the algae Isochrysis sp., Platymonas sp., yeast and photosynthetic bacteria Rhodospirillum sp. Supplemental foods used for hard clam growout ponds include fishmeal, fish soluble, soybean meal, commercial formulated food and other home made mixtures in powder form. Clam farmers develop their diets and feeding strategies, such as the amount and timing of food delivery and assess the feeding effects on environmental quality completely based on their own experience, without using the available information from scientific studies. Therefore, this study aims to determine the effects of various concentrations of six diets that are currently or could potentially be used for hard clam aquaculture, on filtration rate and pseudofeces production of hard clams of different sizes so that this basic information can be used to develop compound feed formulation and appropriate feeding strategies.

MATERIALS AND METHODS

Experimental Variables: Diets, Their Concentrations and Clam Sizes

The 6 experimental diets evaluated were fishmeal (F), soybean meal (S), commercial hard clam formulated feed (C), live microalgae: Tetraselmis chui (T) and Nannochloropsis oculata (N) and bread yeast (Y). The major ingredients in C are S, F, Y, squid meal, corn meal, oyster shell, yeast and calcium perphosphate. The algae T and N were often found in hard clam ponds during our field observations and have been widely used as feed for bivalves (Laing & Gil Verdugo t991, Laing & Millican 1992). The feed components F, S, Y and C were obtained from a commercial source (Taishan Co., Salu, Taichuan, Taiwan). Microalgal stock cultures were obtained from Biotechnology Section, Taiwan Fisheries Research Institute. These algae were cultured in 5-L glass flasks containing Provasoli's enriched seawater (35 [per thousand] salinity, 0.45 [micro]m filtered and UV treated) (Provasoli 1968). Cultures were held at 25[degrees]C under cool white fluorescent light on a 12 hL: 12 hD photoperiod. Algae were used during mid stationary phase of growth. The diet F and S were further ground by a locally made feed ingredient grinder for 4 min and 8 min, respectively to obtain finer particles sizes. Particle sizes were determined by measuring at least 300 diet particles to the nearest 5 p,m with a hemacytometer under a microscope (Nikon E400, Tokyo, Japan). Intervals were set: <5 [micro]m, every 5 [micro]m from 6-30 [micro]m and >30 [micro]m. Particle sizes (mean maximum length [+ or-] standard deviation) and shape (determined qualitatively) of the experimental diets were: F = 17.3 [+ or -] 7.5 [micro]m, irregular; S = 19.5 [+ or -] 10.2 [micro]m, irregular; C = 12.5 [+ or -] 6.3 [micro]m, irregular; T = 8.0 [+ or -] 1.5 [micro]m, oval; n = 4.1 [+ or -] 0.7 [micro]m, round and Y = 6.2 [+ or -] 1.0 [micro]m, round.

The diet concentrations used were determined by using the maximum concentration of N. oculata that could be cultured in this laboratory, around 2,000 mg dry biomass [L.sup.-1], as 2 units in a [log.sub.10] scale. We then chose 0.5, 1 and 1.5 units as the concentrations for use, which were equivalent to 20 mg, 200 mg and 633 mg dry weight [L.sup.-1]. In clam ponds studied, algal concentration could reach as much as 435 mg dry weight [L.sup.-1] depending on their productivity.

Dry weights of algal cells of both species were determined by filtering algae from a 100-mL aliquot of suspension of known concentration; aliquots were taken from five replicate cultures of each species. Algae were retained on tared, glass-fiber filters (Whatman, no.GF/F, 0.7-[micro]m pore size), which were subsequently washed with a 0.5 M solution of ammonium formate to remove sea salts. Filters were then dried at 100[degrees]C for 2 h to volatilize the ammonium formate (Epifanio & Ewart 1977) and weighed on an analytical balance to a precision of 0.1 mg. Algal density in dry weight base were 1.18 ([+ or -] 0.43) x [10.sup.8] cells [mg.sup.-1] and 1.85 ([+ or -] 0.67) x [10.sup.7] cells [mg.sup.-1] for N and T, respectively.

Three size classes of clam were used: small, 7-11.9 g (total body wet weight); medium, 12-17.9 g and large, 18-26 g. Clams were obtained from ponds in Taishi station, Mariculture Research Center, Taiwan Fisheries Research Institute. At our laboratory, they were acclimated for a week under ambient temperature in a 2,000-L fabricated reinforced polyethylene (FRP) round tank paved with 6-10 cm sand, and fed a combination of all 6 diets on an equal dry weight basis. Salinity was maintained at 15%o to 18 [per thousand], which was the salinity in the ponds where the clams were from. One day before experimentation, an adequate number of clams were collected and placed in a 20-L bucket filled with 1-[micro]m filtered seawater. Water temperature was controlled at around 25.2[degrees]C [+ or -] 0.4[degrees]C and clams were not fed.

Standard Curve of Suspended Particles

A turbidity-weight relationship was developed to estimate the biomass of each diet in the water. Five concentrations: 20 mg, 63.3 mg, 200 mg, 633 mg and 2,000 mg [L.sup.-1] of F, S, C and Y suspension were obtained by weighing 0.012, 0.038, 0.12, 0.38 and 1.2 g of each diet into a beaker containing 600 mL filtered seawater and suspended with a magnetic stirrer. A 10-mL sample was taken by pipette and immediately measured in a turbidity meter (HACH 2100P, Loveland, Colorado, USA). For each concentration, 5 replicates were measured and for each replicate, 2 repeated readings were obtained. For the microalgae, instead of dilution, the original algal suspension was concentrated several times by centrifugation at x3,000 rpm for 10 min each time to obtain a calibration.

Filtration Rate Apparatus

Clearance rates were determined using methods similar to those of Shumway et al. (1985) and Levinton et al. (2002). Filtration rate was determined by the indirect method (Epifanio & Ewart 1977, Winter 1978), measuring the removal of suspended particles from a known volume of water per unit time (Fox et al. 1937). This "indirect method" agrees well with other indirect and direct methods used for measuring filtration rates in mussels (Famme et al. 1986). The feeding apparatus was composed of a 1-L beaker held over a magnetic stirrer (MS-90, Fargo). The beaker contained a feeding platform, set above a 3-cm Teflon stirrer, made by tying three 3.5-cm rods perpendicularly to a 6-cm ring. Both ring and rods were made of no. 19 gauge (1.9 mm inner diameter) insulated wire. The ring was covered with a 2 mm x 2 mm mesh nylon net. The dial of the stirrer was fixed at 3.5 so that mixing kept all diet particles in suspension but would not resuspend the clam's fecal material. One clam was used per beaker.

Each trial was conducted as follows: a designated diet suspension was prepared and 600 mL of diet suspension was poured into a 1-L beaker. The stirrer was activated and a designated size clam, which had been starved for 24 h, was placed on the center of the feeding platform. A 10-mL sample of suspension was collected to measure the initial diet concentration. The experiment ran for 1 h starting when the clam resumed feeding and extended and dilated its siphon. Another sample was taken at the end of the hour for the final diet concentration. In a few preliminary trails, when large size clams were used to test their filtration capability in diet F and S at a concentration 20 mg [L.sup.-1] for 10 and 15 min, no significant reduction in diet concentration were found. In a similar clearance experiment, Levinton et al. (2002) used a clearance period of 0.75 h for mussels and 1.5 h for oysters, thus we chose 1 h for each trial. Experimental bivalves were then opened; their soft tissue was separated from the shell, blotted with a paper towel and then dried at 105[degrees]C for around 48 h until a constant weight was reached. Dry weight data were used to calculate weight specific filtration rate.

Considering the deviation of experimental conditions from the natural ones, such as starvation prior to the measurement, constant circular flow around the clam and clam's laying position on the feeding platform, filtration rate determined by this indirect method may not be very representative of the true filtration rate of this hard clam in natural conditions. However, for the simplicity of our method, apparatus set-up and the consistency in use, the filtration rate should potentially be used in a comparative manner among treatments.

Filtration Rate Experiment

A 3 x 6 x 3 factorial arrangement treatments design was used in which three sizes of clam were exposed to six diets each at three concentrations. Each treatment combination had four replicates. Each replicate used 3 feeding chambers: a blank chamber using a dummy clam and two treatment chambers with live clams. The dummy clam consisted of an empty clam filled with sand with the shells glued together. In each replicate the clams had a similar shell size. The experiments in three chambers of a replicate were run simultaneously. After correcting by turbidity changes in the blank chamber, the two readings for live clams were averaged and used as one datum for that replicate.

The equation by Riisgard (1988) was used to estimate the filtration rate (F):

F = V/t x ln Co/C+t

where V is a known volume of water; t the time in h; In the natural logarithm; and Co and Ct the particle concentrations at time 0 and t; respectively. Because the rate of filtration of bivalves is related to the size of the animal (Winter 1978) and clam weight varied for each trial, weight-specific filtration rate (FR = F/W in mL [h.sup.-1] [g.sup.-1]) (Epifanio & Ewart 1977) was used: where W was dry weight of soft tissue mass (g) (Bayne et al. 1976).

Pseudofeces Production Experiment

A factorial treatments design: diets 6 x 2 clam sizes were used in which each treatment had four replicates. Only 1 diet concentration was used, 200 mg [L.sup.-1]. This was because some of the clams would not feed at 633 mg dry weight [L.sup.-1]. On the other hand, at the lowest concentration, 20 mg [L.sup.-1], insufficient pseudofeces could be collected to allow accurate measurement. No medium size clams were available when conducting the experiment so only small and large clams were used.

After feeding for 1 h, the clam was removed from the beaker. A pipette was used to carefully collect the pseudofeces on the bottom or attached to the wall of the beaker or that clung to the feeding platform. They were placed in an aluminum foil plate (5 cm in diameter and about 1.9 g weight), dried to constant weight at 80[degrees]C and weighed to the nearest 0.01 mg.

Statistical Analysis

For filtration rate, a 3-way ANOVA was used to test the significance of the main effects and interactions of diets, diet concentrations and clam sizes. For pseudofeces production, a 2-way ANOVA was used to test the significance on the main effects and interactions of diets and clam sizes. Duncan's multiple range tests (DMRT) were used to test the differences among the levels of each main effect. Besides DMRT for pairing (one-to-one) comparisons, 5 orthogonal contrasts were conducted for systematic (set-to-set) comparisons: (1) diet particle shape: regular (N, T and Y) versus irregular (C, F and S); (2) regular particle shape: algae (N and T) versus yeast (Y); (3) algae: N versus T; (4) irregular particle shape: mixture components (C) versus single component (F and S) and (5) single component: plant material (S) versus animal material (F).

RESULTS

Filtration Rate

There were highly significant (P [less than or equal to] 0.01) effects of diet, diet concentrations and clam sizes on filtration rate, and all the interactions were highly significant (Table 1). Within diet, orthogonal contrasts (Table 1) indicated that the average filtration rate for diets having regular shape of particles (i.e., N, T and Y) was significantly lower than that for diets having an irregular particle shape (i.e., C, F and S). For the regular particle shape diets, average filtration rate for the planktonic algae N and T was significantly lower than that for the yeast, Y. Between the two algae, clams fed N had significantly higher filtration rates than those fed T. For the irregular particle shape diets, filtration rate for the diet having mixed components (i.e., C) was significantly lower than the average filtration rate for diets having a single component F and S. Between the diets that had a single component, clam filtration rate of S was significantly higher than that of F. Disregarding clam sizes, DMRT results showed that the filtration rates in descending order were S > C > F = Y > N > T (Fig. 2). For small clams, all differences in filtration rate among diets were significant (i.e., S > C > F > Y > N > T). However, for larger clams, the differences in filtration rate among diets became less distinct. For example, for large clams, there were no differences in filtration rate between S and F, and also no differences among S, Y and C.

[FIGURE 2 OMITTED]

Overall, filtration rate decreased with increasing diet concentration (Fig. 3). Such an inverse relationship was especially evident between diet concentration 633 mg [L.sup.-1] and 200 mg [L.sup.-1] and this effect was consistent for each clam size class (i.e., whereas the former diet concentration was about 3 times as the latter, the filtration rate at the former was about 1/3 of the latter).

[FIGURE 3 OMITTED]

Disregarding diet and diet concentration, the smaller the clam, the higher the weight-specific filtration rate. The filtration rates differed significantly and were 49 [+ or -] 2, 30 [+ or -] 1 and 27 [+ or -] 1 mL [h-.sup.-1] [g.sup.-1] for small, medium and large clams, respectively.

Pseudofeces Production

There were highly significant effects of diet and clam size on pseudofeces production. No interaction was found between diet and clam size on pseudofeces production (Table 2). Overall, large clams produced significantly less pseudofeces (1.3 [+ or -] 0.9 mg [g.sup.-1]) than small clams (1.7 [+ or -] 0.8 mg [g.sup.-1]).

Within the diet effects, orthogonal contrasts (Table 2) indicated that the average pseudofeces production from diets having a regular particle shape (i.e., N, T and Y) did not differ from that of diets having an irregular particle shape (i.e., C, F and S). For the diet with a regular particle shape, average pseudofeces production from the two planktonic algae N and T, was significantly lower than that from the yeast Y. Clams fed on N produced significantly less pseudofeces than those fed on T. For the diet with irregular particle shape, pseudofeces production from the mixed diet (i.e., C) was significantly lower than that from single-component diets (i.e., F and S). Between the single-component diets, the pseudofeces production from S was not different from that of F. Among-diet effects showed that for clams of both sizes (Fig. 4), those fed on Y produced the highest amount of pseudofeces. Among the remaining diets, pseudofeces production descended in the following order: S [greater than or equal to] F [greater than or equal to] T [greater than or equal to] C [greater than or equal to] N. Pseudofeces production for F, T and C did not differ significantly. When DMRT was performed by clam size separately, the order for large or small size classes remained the same as for both sizes combined. The only difference in DMRT results between large and small clams was that for the latter Y was not significantly greater than S but for the former pseudofeces production differed significantly between these two diets.

[FIGURE 4 OMITTED]

DISCUSSION

In general, suspension-feeding bivalve molluscs have adopted several strategies for controlling the ingestion of particulate matter (MacDonald & Ward 1994), including regulation of (1) feeding duration (Foster-Smith 1975); (2) clearance rates (Bayne & Newell 1983) and (3) pseudofeces production (Kiorbce et al. 1980, Newell & Jordan 1983). Various criteria are often proposed as the basis for particle selection including physical features: such as particle size (Tammes & Dral 1955, Mohlenberg & Riisgard 1978, Riisgard 1988, Langdon & Newell 1990), shape (Bayne et al. 1977), aggregation (Waite et al. 1995), motility and density (Brillant & MacDonald 2000), chemical cues: such as energy content, C/N ratio (Ward & MacDonald 1996), organic content (Bacon et al. 1998, Defossez & Hawkins 1997) and chlorophyll content (Nakamura 2001). The production of pseudofeces can serve to improve the quality of material ingested by means of selective rejection, rather than simply the elimination of excess material of the ingestive capacity (MacDonald & Ward 1994). Selective rejection mainly involves preferential rejection of nonchlorophyll-containing particles (Kiorboe & Mohlenberg 1981, Newell & Jordan 1983) and reduction of the proportion of particulate inorganic matter in pseudofeces (Widdows et al. 1979, Kiorboe et al. 1980, Kiorboe & Mohlenberg 1981, Bricelj & Malouf 1984, Defossez & Hawkins 1997).

Diets

The highest filtration rate obtained for soybean meal in this study (Fig. 2) could be mainly attributed to its having the largest particle size, which made S easier to be retained by the gill of clam and left less particles in water. Previous studies have demonstrated the importance of particle size in mediating selection processes in some bivalve species (Defossez & Daguzan 1996, Defossez & Hawkins 1997). Wisely and Reid (1978) considered the selection of feed by members of the order Eullamellibranchia (oysters, cockles and clams); selection appeared to be made on the basis of particle size regardless of potential food value. In Mytilus edulis, Cerastoderma edule and Venerupis pullastra, the rates of ingestion of particles were found to be roughly proportional to the size of the particles (Foster-Smith 1975). A few studies indicated that bivalves had higher filtration or retention efficiency for larger particles. The filtration efficiency of bacterioplankton (0.2-2 [micro]m) by Geukensia demissa was 42% lower that that of phytoplankton >2 [micro]m (Wright et al. 1982). Mercenaria mercenaria completely retained particles above 4 [micro]m. Below this size threshold retention efficiency gradually decreased to between 35% and 70% for 2-[micro]m particles (Riisgard 1988). In the clam Ruditapes decussatus particles smaller than 3 [micro]m in diameter, which include bacteria and clay particles, were retained with low efficiency (i.e., <75%). Algal cells, such as phytoplankton and other particles in the size range 3-8 [micro]m were efficiently retained (70% to 100% retention) by the clam (Sobral & Widdows 2000). The studies mentioned earlier concluded that filtration retention efficiency increased with increasing particle size from 0.2-8 [micro]m. In our study, because the particle size of diets varied over a wider range, 4 [micro]m to 19 [micro]m, the overall ranking of filtration rates for the diets (S > C > F = Y > N > T, Fig. 2) did not completely follow the ranking of particle size: S (19 [micro]m) > F (17 [micro]m) > C (12 [micro]m) > T (8 [micro]m) > Y (6 [micro]m) > N (4 [micro]m). Bacon et al. (1998) indicated that because most of the particles used in their study were between 4 and 6 [micro]m in diameter, size-dependent selection was probably not a significant factor and selection by the softshell clam M. arenaria was based on organic content, or perhaps other qualitative particle characteristics.

That particle size that can affect pseudofeces production is indicated by the comparison of the two algae T and N; the higher pseudofeces production when clams were fed on T may be attributed to the fact that T particles are twice as big as N particles or to other confounding factor. Similarly, Defossez and Hawkins (1997) experimented with the mussel Mytilus edulis and clams Ruditapes philippinarum and Tapes decussates and they concluded that particles with diameters larger than from between 7.5 and 22.5 [micro]m were preferentially rejected as pseudofeces. As with filtration rate, particle size was not the only factor affecting pseudofeces production. In our study, pseudofeces production in descending order was Y > S [greater than or equal to] F [greater than or equal to] T [greater than or equal to] C [greater than or equal to] N (Fig. 4), which again did not follow completely the order of particle size. The result that particle selection via pseudofeces production was not totally based on the size, was in agreement with MacDonald and Ward (1994).

The results of the orthogonal contrast indicated that the average filtration rate for diets with a more regular particle shape (i.e., N, T and Y) was significantly lower than that for diets consisting of irregular particle shape (i.e., C, F and S). Few studies have been published on the effects of shape regularity of diet particles on filtration rate in bivalves. Bayne et al. (1977) pointed out that elongated or triradiated algal cells might be more efficiently retained than spherical particles of the same volume. Wisely and Reid (1978) mentioned that rice particles that were used in their study, when viewed microscopically, were mainly irregularly shaped but many of them had a characteristic "hexagonal" appearance. Irregular shape may not only extend particle size in one dimension but the rough extrusions may also favor the retention of particles by the gill and consequently reduce the particle density in water in this study.

Limited studies showed that a diet of 50/50 mixture of algae and yeast supported growth comparable to a 100% algal ration when fed to the hard clam Mercenaria mercenaria (Epifanio 1979) and oyster Crassostrea virginica (Alatalo 1980). Although yeast could be used as algal substitutes for feeding bivalves, filtration rate or ingestion selectivity was not compared between yeast and algae in those studies. In our study, filtration rate and pseudofeces production were higher for yeast Y than for planktonic algae N and T grouped together. The higher filtration rate for Y could not be attributed to particle size, because particle size of Y (6 [micro]m) was between T (8 [micro]m) and N (4 [micro]m). Nonetheless particles above 4 Ixm can be completely retained by Mercenaria mercenaria (Riisgard 1988). Higher pseudofeces production in Y for this hard clam might be attributed to yeast's lack of chlorophyll, because it was suggested that one of the two ways in which scallops could improve the quality of material ingested was through preferential rejection of nonchlorophyll-containing particles in the pseudofeces (Kiorboe & Mohlenberg 1981, Newell & Jordan 1983).

Filtration rates of clams fed on C were higher than when they were fed on F, Y and N, which were larger in particle size. This could be attributed to the hard clam's greater acceptance of C, which is possibly closer to a natural suspension comprising a mixture of various components than the other diets that contain only a single component. Previous studies demonstrated that addition of a small amount of mud to an algal suspension made the suspension closer to a natural one and only improved the clearance rate for Mytilus edulis (Kiorboe et al. 1980, Bayne et al. 1987) and V. corrugatus (Stenton-Dozey & Brown 1994).

Bivalves are traditionally regarded as suspension feeders for mainly phytoplankton and organic particles. In our study, pseudofeces production for F was no greater than S suggesting that for this clam preference for fishmeal, an animal-based substance, was no less than soybean meal, a plant-based substance. The higher filtration rate for S than for F could be attributed to its being 2 [micro]m larger than the latter, or better quality.

Diet Concentrations

Our findings that filtration rate in Meretrix lusoria decreased with increasing concentration for all diets (Fig. 4) were in agreement with other literature studies for Mytilus edulis, Cerastoderma edule and Venerupis pullastra, filtering purely algal suspensions (Foster-Smith 1975); for Mytilus edulis feeding on resuspended fine mud (Widdows et al. 1979); for Ostrea edulis (Grant et al. 1990), for Mya arenaria (Grant & Thorpe 1991) and for Cerastoderma edule (Iglesias et al. 1992, Navarro & Widdows 1997), all feeding on mixtures of algal cells and suspended silt; and for Ruditapes decussatus, filtering suspended particulate matter (Sobral & Widdows 2000).

Bivalves can typically maintain a constant ingestion when exposed to increasing seston concentrations by reducing clearance rate, increasing pseudofeces production or some combination of both mechanisms (Foster-Smith 1975, Winter 1978, Kiorboe et al. 1980, Bricelj & Malouf 1984, Bacon et al. 1998).

Filter-feeding activity is a function of cell concentration, which has been well documented by several authors for different species of lamellibranchiate bivalves (Sania 1976, Epifanio & Ewart 1977, Winter 1978). Similar reductions in clearance rates in response to increasing particle concentrations have been observed in epifaunal bivalves (Bayne et al. 1987) and infaunal bivalves including Mercenaria mercenaria (Bricelj & Malouf 1984). Mya arenaria significantly decreases its clearance rate in response to increasing particle concentration, showing about a 50% decline in rates, especially between 1 and 7 mg [L.sup.-1] (Bacon et al. 1998). In our study, although diet concentrations were much higher, ranging between 20 mg [L.sup.-1] and 633 mg [L.sup.-1], the decrease in filtration rate still held.

Clam Sizes

There have been few studies on body size effects on filtration rate in tropical marine bivalves, such as the species used in this study. Our results showed that weight-specific filtration rate declined with increase in body size (Fig. 1, Fig. 2). Instead of using weight-specific filtration rate, Riisgard (1988) demonstrated that the filtration rate (F, L [h.sup.-1]) in Mercenaria mercenaria, Crassostrea virginica and Geukensia demissa increased with increasing dry weight of soft parts (W, g) according to the equation: F = a [W.sup.b]. Such allometric relationship was well applied to several bivalves of which the filtration rates were measured by various methods (Gosling 2003). Nakamura (2001) also showed that the clearance rate for each category of chlorophyll a-containing particles (bacteria, picocyanobacteria, flagellates and Nitzschia) had a positive correlation with soft-body dry weight. In fact, there was no contradiction in results between those and ours, because weight-specific filtration rate or unit weight filtration rate (Epifanio & Ewart 1977) was inversely related to the weight of the animal. That the rates of physiological processes increase as power relationships with increasing body size, but rates per unit body mass tend to decrease with increasing size (Yukihira et al. 1998) provided further explanation for the relationship between filtration rate or weight-specific filtration rate and weight. Disregarding diet type, diet concentration and size class the allometric relationship between filtration rate and weight in this study was F = 6.95 [W.sup.0.81].

Interaction

This study showed that the interactions between clam sizes, diets and diet concentrations had significant effects on filtration rate. It was obvious that the acceptance of various diets by small clams was different from that in large clams, if filtration rate was used as a measure of diet acceptance. Such interaction effects on filtration rate, to our surprise, were almost never reported. The only related study by Perez-Camacho et al. (1994), indicated that the filtration rate of Ruditapes decussatus veliger larvae decreased with increasing diet concentration; however, the decrease in filtration rate was less pronounced in large than small clams. Such dissimilar responses for animals of different sizes were similar to those obtained in our study.

This study used filtration rate and pseudofeces production as response parameters to determine the ingestion preference of various sizes of hard clams for diets of various categories and concentrations. We first used DMRT to examine the feasibility of using particle size as an individual effect to explain ingestion preference across all 6 diets. Furthermore, we used orthogonal contrasts to compare systematically the ingestion preference for diets under various categories, such as shape regularity of diet particle (regular vs. irregular), chlorophyll content in live microbes (phytoplankton vs. yeast), complexity of diet composition (single component vs. multiple components) and plant-based diet versus fishmeal. The results suggested the complications in ingestion preference (Ward & Shumway 2004) require many more specific and detailed trials before a practical and realistic compound feed for the hard clam can be formulated.

LITERATURE CITED

Alatalo, P. 1980. Yeast utilization in oysters and clams. Master's thesis, University of Delaware.

Bacon, G. S., B. A. MacDonald & J. E. Ward. 1998. Physiological responses of infaunal (Mya arenaria) and epifaunal (Placopecten magellanicus) bivalves to variations in the concentration and quality of suspended particles. I. Feeding activity and selection. J. Exp. Mar. Biol. Ecol. 219:105-125.

Bayne, B. L. & R. C. Newell. 1983. Physiological energetics of marine mollusks. In: K. M. Wibur & A. S. Saleuddin, editors. The mollusca, Vol. 4. New York: Academic Press. pp. 407-515.

Bayne, B. L., A. J. S. Hawkins & E. Navarro. 1987. Feeding and digestion by the common mussel Mytilus edulis L. (Bivalvia: Mollusca) in mixture of silt and algal cells at low concentration. J. Exp. Mar. Biol. Ecol. 111:1-22.

Bayne, B. L., R. J. Thompson & J. Widdows. 1976. Physiology: I. In: B. L. Bayne, editor. Marine mussels: their ecology and physiology. Cambridge: Cambridge University Press. pp. 121-206.

Bayne, B. L., J. Widdows & R. I. E. Newell. 1977. Physiological measurements on estuarine bivalve molluscs in the field. In: B. F. Keegan, et al., editors. Biology of benthic organisms, Pergamon. pp. 57-68.

Bricelj, V. M. & R. E. Malouf. 1984. Influence of algal and suspended sediment concentrations on the feeding physiology of the hard clam Mercenaria mercenaria. Mar. Biol. 84:155-165.

Brillant, M. G. S. & B. A. MacDonald. 2000. Postingestive selection in the sea scallop Placopecten magellanicus (Gmelin): the role of particle size and density. J. Exp. Mar. Biol. Ecol. 253:211-227.

Chen, H. C. 1984. Recent innovations in cultivation of edible mollusks in Taiwan, with special reference to the small abalone Haliotis diversicolor and the hard clam Meretrix lusoria. Aquaculture 39:11-27.

Chen, H. C. & R. Y. Lyuu. 1982. Studies on the artificial propagation of the hard clam, Meretrix lusoria. I. Sex distinction. J. Mar. Sci. 27:1-8.

Coutteau, P., N. Hadley, J. Manzi & P. Sorgeloos. 1991. Manipulated yeast diets as a partial algal substitute for the nursery culture of the hard clam Mercenaria mercenaria. In: Aquaculture and the environment. Aquaculture Europe '91, Dublin, Ireland, June 10-12, 1991, Short communications and abstracts. Eur. Aquacult. Soc. Spec. Public 14. 77-78.

Coutteau, P., P. Lavens, P. Leger & P. Sorgeloos. 1990. Manipulated yeast diets as a partial substitute for rearing bivalve molluscs: laboratory trials with Tapes semidecussata, in: World Aquaculture '90, June 10-14, 1990, Halifax, NS, Canada, Book of Abstracts, IMPRICO, Quebec, Canada. pp. 111.

Defossez, J. M. & J. Daguzan. 1996. About preferential ingestion of organic matter by bivalves. J. Mollus. Stud. 62:394-397.

Defossez, J. M. & A. J. S. Hawkins. 1997. Selective feeding in shellfish: size-dependent rejection of large particles within pseudofeces from Mytilus edulis, Ruditapes philippinarum and Tapes decussatus. Mar. Biol. 129:139-147.

Donaldson, J. 1991. Commercial production of microalgae at Coast Oyster Company. In: W. Fulks & K. L. Main, editors. Rotifer and microalgae culture systems. Proceedings of a US-Asia Workshop, Honolulu, Hawaii, January 28-31, 1991. The Oceanic Institute, Hawaii, pp. 229 236.

Epifanio, C. E. 1979. Comparison of yeast and algal diets for bivalve mollusks. Aquaculture 16:187-192.

Epifanio, C. E. & J. Ewart. 1977. Maximum ration of for the oyster Crassostrea virginica. Aquaculture 11:13-29.

Fisheries Administration 2003. Taiwan Fisheries Statistical Yearbook.

Famme, P., H. U. Riisgard & C. B. Jorgensen. 1986. On direct measurement of pumping rates in the mussel Mytilus edulis. Mar. Biol. 92: 323-327.

Foster-Smith, R. L. 1975. The effect of concentration of suspension on the filtration rates and pseudofaecal production for Mytilus edulis (L), Cerastoderma edule (L) and Venerupis pullastra (Montagu). J. Exp. Mar. Biol. Ecol. 17:1-22.

Fox, D. L., H. N. Sverdrup & J. R. Cunningham. 1937. The rate of water propulsion by the California mussel. Bio. Bull. Woods Hole, Mass. 72:417-438.

Gladue, R. 1991. Heterotrophic microalgae production: potential for application to aquaculture feeds. In: W. Fulks & K. L. Main, editors. Rotifer and microalgae culture systems. Proceedings of a US-Asia Workshop, Honolulu, Hawaii, January 28-31, 1991. The Oceanic Institute, Hawaii. pp. 276-286.

Gosling, E. M. 2003. Bivalve molluscs. Fishing News Books. Oxford, UK: Blackwell Publishing. pp. 443.

Grant, J. & B. Thorpe. 1991. Effects of suspended sediment on growth, respiration and excretion of the soft-shell clam (Mya arenaria). Can. J. Fish. Aquat. Sci. 47:1285-1292.

Grant, J., C. T. Enright & A. Griswold. 1990. Resuspension and growth of Ostrea edulis: a field experiment. Mar. Biol. 104:51-59.

Guo, Z. J. 2003. Adjustment and strategies responding to low price stage of hard clam culture industry. Fish. Ext. 201:13-29. (in Chinese).

Ho, Y. D. 1991. Growth of the hard clam, Meretrix lusoria cultured in ponds in Taiwan. J. Fish. Soc. Taiwan 18:273-277.

Iglesias, J. I. P., E. Navarro, P. Alvarez Jorna & I. Armentia. 1992. Feeding, particle selection and absorption in cockles Cerastoderma edule (L.) exposed to variable conditions of food concentration and quality. J. Exp. Mar. Biol. Ecol. 162:177-198.

Jones, D. A., D. L. Holland & S. S. Jabborie. 1984. Current status of microencapsulated diets for aquaculture. Appl. Biochem. Technol. 10: 275-288.

Kiorboe, T. & F. Mohlenberg. 1981. Particle selection in suspension-feeding bivalves. Mar. Ecol. Prog. Ser. 5:291-296.

Kiorboe, T., F. Mohlenberg & O. Nohr. 1980. Feeding, particle and selection and carbon absorption in Mytilus edulis in different mixtures of algae and resuspended bottom material. Ophelia 19:193-205.

Laing, I., A. R. Child & A. Janke. 1990. Nutritional value of dried algae diets for larvae of manila clam (Tapes philippinarum). J. Mar. Biol. Ass. U.K. 70:1-12.

Laing, I. & P. F. Millican. 1992. Indoor nursery cultivation of juvenile bivalve mollusks using diets of dried algae. Aquaculture 102:231-243.

Laing, I. & C. Gil Verdugo. 1991. Nutritional value of spray-dried Tetraselmis suecica for juvenile bivalves. Aquaculture 92:207-218.

Langdon, C. J. & R. 1. E. Newell. 1990. Utilization of detritus and bacteria as food sources by two bivalve suspension-feeders, the oyster Crassostrea virginica and mussel Geukensia demissa. Mar. Ecol. Prog. Ser. 58:299-310.

Langdon, C. J., D. M. Levine & D. A. Jones. 1985. Microparticulate feeds for marine suspension-feeders. J. Microencapsul. 2:1-11.

Levinton, J. S., J. E. Ward & S. E. Shumway. 2002. Feeding responses of the bivalves Crassostrea gigas and Mytilus trossulus to chemical composition of fresh and aged kelp detritus. Mar. Biol. 141:367-376.

MacDonald, B. A. & J. E. Ward. 1994. Variation in food quality and particle selectivity in the sea scallop Placopecten magellanicus (Mollusca; Bivalvia). Mar. Ecol. Prog. Ser. 108:251-264.

Mohlenberg, F. & H. U. Riisgard. 1978. Efficiency of particle retention in 13 species of suspension feeding bivalves. Ophelia 17:239-246.

Nakamura, Y. 2001. Filtration rates of Manila clam, Ruditapes philippinarum: dependence on prey items including bacteria and picocyanobacteria. J. Exp. Mar. Biol. Ecol. 266:181-192.

Navarro, J. M. & J. Widdows. 1997. Feeding physiology of Cerastoderma edule in response to a wide range of seston concentrations. Mar. Ecol. Prog. Ser. 152:175-186.

Newell, R. I. & S. J. Jordan. 1983. Preferential ingestion of organic material by American oyster Crassostrea virginica. Mar. Ecol. Prog. Ser. 9:203-223.

Perez-Camacho, A., R. Beiras & M. Albentosa. 1994. Effect of algal food concentration and body size on the ingestion rates of Ruditapes decussatus (Bivalvia) veliger larvae. Mar. Ecol. Prog. Ser. 115:87-92.

Provasoli, L. 1968. Media and products for the cultivation of marine algae. In: A. Watanabe & A. Hattori, editors. Culture and collections of algae. Tokyo: Japanese Society of Plant Physiology. pp. 63-75.

Riisgard, H. U. 1988. Efficiency of particle retention and filtration rate in 6 species of Northeast American bivalves. Mar. Ecol. Prog. Ser. 45: 217-223.

Sania, L. V. 1976. Rate and intensity of filtration in some Caspian Sea bivalve molluscs. Oceanology (USSR) 15:496-498.

Shumway, S. E., T. L. Cucci, R. C. Newell & C. M. Yentsch. 1985. Particle selection, ingestion, and absorption in filter-feeding bivalves. J. Exp. Mar. Biol. Ecol. 91:77-92.

Sobral, P. & J. Widdows. 2000. Effects of increasing current velocity, turbidity and particle-size selection on the feeding activity and scope for growth of Ruditapes decussatus from Ria Formosa, southern Portugal. J. Exp. Mar. Biol. Ecol. 245:111-125.

Southgate, P. C., A. C. Beer, P. F. Duncan & R. Tamburri. 1998. Assessment of the nutritional value of three species of tropical microalgae, dried Tetraselmis and a yeast-based diet for larvae of the blacklip pearl oyster, Pinctada margaritifera (L.). Aquaculture 162:247-257.

Stenton-Dozey, J. M. E. & A. C. Brown. 1994. Short-term changes in the energy balance of Venerupis corrugatus (Bivalvia) in relation to tidal availability of natural suspended particles. Mar. Ecol. Prog. Ser. 103: 57-64.

Tammes, P. M. L. & A. D. G. Dral. 1955. Observations of the straining of suspensions by mussels. Arch. Meerl. Zool. 11:87-112.

Urban, E. R. & C. J. Langdon. 1984. Reduction in costs of diets for the American oyster, Crassostrea virginica (Gmelin), by the use of non-algal supplements. Aquaculture 38:277-291.

Waite, A. M., R. J. Olson, H. G. Dam & U. Passow. 1995. Sugar containing compounds on the cell surfaces of marine diatoms measured using concanavalin A and flow cytometry. J. Phycol. 31:925-933.

Ward, J. E. & B. A. MacDonald. 1996. Pre-ingestive feeding behaviors of two sub-tropical bivalves (Arca zebra and Pinctada imbricata): responses to an acute increase in suspended sediment concentration. Bull. Mar. Sci. 59:417-432.

Ward, J. E. & S. E. Shumway. 2004. Separating the grain from the chaff: particle selection in suspension- and deposit-feeding bivalves. J. Exp. Mar. Biol. Ecol. 300:83-130.

Widdows, J., P. Fieth & C. M. Worrall. 1979. Relationships between seston available food and feeding activity in the common mussel Mytilus edulis. Mar. Biol. 50:195-207.

Winter, J. E. 1978. A review on the knowledge of suspension-feeding in lamellibranchiate bivalves, with special reference to artificial aquaculture systems. Aquaculture 13:1-33.

Wisely, B. & B. L. Reid. 1978. Experimental feeding of Sydney rock oysters (Crassostrea commercialis = Saccostrea cucullata). I. Optimum particle sizes and concentrations. Aquaculture 15:319-331.

Wright, R. T., R. B. Coffin, C. P. Ersing & D. Pearson. 1982. Field and laboratory measurements of bivalve filtration of natural marine bacterioplankton. Limnol. Oceanogr. 27:91-98.

Yukihira, H., D. W. Klumpp & J. S. Lucas. 1998. Effect of body size on suspension feeding and energy budgets of the pearl oysters Pinctada margaritifera and P. maxima. Mar. Ecol. Prog. Ser. 170:119-130.

YEW-HU CHIEN * AND WEN-HUA HSU 

Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan 202, Republic of China

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

TABLE 1.

Main effects of 6 diets, 3 diet concentrations, 3 clam sizes, their
interactions and 5 orthogonal contrasts for diets on filtration rate
of hard clam.

              SV                    Mean         df          SS

Model                                            53        125,313
  Diet                                            5         13,948
    Shape (Reg. vs. Irreg.)
      (N, T, Y vs. C, F, S)       29 vs. 42       1          8,930
    Reg. (Algae vs. Yeast)
      (N, T vs. Y)                26 vs. 36       1          2,280
    Algae (Nano. vs. Tetra.)
      (N vs. T)                   27 vs. 24       1            137
    Irreg. (Mixture vs. Single)
      (C vs. F, S)                40 vs. 43       1            173
    Single (Soy. vs. Fish.)
      (S vs. F)                   49 vs. 37       1          2,428
  Clam size                                       2         20,376
  Clam size x Diet                               10          5,402
  Conc.                                           2         51,127
  Clam size x Conc.                               4          2,074
  Diet x Conc.                                   10         11,689
  Clam size x Diet x Conc.                       20         10,697
Error                                           162          5,538
Total                                           215        130,851

              SV                     MS           F         P > F

Model                               2,364      69           <0.01
  Diet                              2,789      81           <0.01
    Shape (Reg. vs. Irreg.)
      (N, T, Y vs. C, F, S)         8,930      16.04        <0.01
    Reg. (Algae vs. Yeast)
      (N, T vs. Y)                  2,280       4.10        <0.01
    Algae (Nano. vs. Tetra.)
      (N vs. T)                       137       0.25        <0.05
    Irreg. (Mixture vs. Single)
      (C vs. F, S)                    173       0.31        <0.05
    Single (Soy. vs. Fish.)
      (S vs. F)                     2,428       4.36        <0.01
  Clam size                        10,188     298           <0.01
  Clam size x Diet                    540      16           <0.01
  Conc.                            25,563     748           <0.01
  Clam size x Conc.                 3,018      88           <0.01
  Diet x Conc.                      1,169      34           <0.01
  Clam size x Diet x Conc.            535      16           <0.01
Error                                  34
Total

TABLE 2. Main effects of 6 diets, 2 clam sizes, their interactions and
5 orthogonal contrasts for diets on hard clam pseudofeces production.

              SV                       Mean           df          SS

Model                                                 11         27.48
  Diet                                                 5         24.69
    Shape (Reg. vs. Irreg.)
      (N, T, Y vs. C, F, S)       1.42 vs. 1.39        1          0.49
    Reg. (Algae vs. Yeast)
      (N, T vs. Y)                0.95 vs. 2.37        1         20.15
    Algae (Nano. vs. Tetra.)
      (N vs. T)                   0.73 vs. 1.17        1          0.79
    Irreg. (Mixture vs. Single)
      (C vs. F, S)                0.90 vs. 1.65        1          2.97
    Single (Soy. vs. Fish.)
      (S vs. F)                   1.78 vs. 1.51        1          0.28
  Clam size                                            1          1.49
  Clam size x Diet                                     5          1.30
Error                                                 36          5.98
Corrected Total                                       47         33.46

              SV                        MS         F       P > F

Model                                  2.50      15.04     <0.01
  Diet                                 4.94      29.73     <0.01
    Shape (Reg. vs. Irreg.)
      (N, T, Y vs. C, F, S)            0.49       2.96      0.09
    Reg. (Algae vs. Yeast)
      (N, T vs. Y)                    20.15     121.34     <0.01
    Algae (Nano. vs. Tetra.)
      (N vs. T)                        0.79       4.75      0.04
    Irreg. (Mixture vs. Single)
      (C vs. F, S)                     2.97      17.89     <0.01
    Single (Soy. vs. Fish.)
      (S vs. F)                        0.28       1.69      0.20
  Clam size                            1.49       9.00     <0.01
  Clam size x Diet                     0.26       1.57      0.19
Error                                  0.17
Corrected Total