The evaluation of spray-dried microalgae in diets for juvenile Manila clam, Tapes philippinarum.

ABSTRACT Replacement of live microalgal diets is a significant challenge in developing cost-effective and reliable production methods for bivalves. In this study, the results of growth experiments are reported with juvenile Manila clams, Tapes philippinarum, fed on a heterotrophically grown, spray-dried, Schizochytrium-based product (SZ; Docosa Gold, Sanders Brine Shrimp) or a spray-dried, Haematococcus pluvialis-based product (H; Sanders Brine Shrimp) either alone or as supplements for rations of living algae. The effects of additions of bentonite, a naturally occurring silt, in combination with SZ and H on the growth of Manila clams were also investigated. Results showed that SZ and H supported clam growth when added as supplements to rations of a mixed diet of the live algae Isochrysis galbana and Chaetoceros calcitrans. Increases in clam wet weight with a 1/4 ration of live algae and a supplement of a full ration (equivalent to the dry weight of the full algal ration) of SZ or H were similar to those of clams fed on a full live algal ration alone. The results indicated that up to 75% substitution of live algae is possible using heterotrophically grown, spray-dried SZ or H without significantly reducing the live (wet) growth of the clams. No significant differences in clam growth rate (% wet and organic weight increase) were found when bentonite was added to diets containing different concentrations of H.

KEY WORDS: Manila clam; spray-dried algae; Haematococcus pluvialis; Schizochytrium sp.; bentonite, Tapes philippinarum

INTRODUCTION

The Manila clam, Tapes philippinarum, is a commercially important, worldwide cultured species. The traditional nursery culture of juvenile Manila clams continues to rely on large-scale production of live microalgae. However, costs of algal production are high for rearing bivalves under nursery conditions, ranging from $160 to more than $200 per kg dry weight (Coutteau & Sorgeloos 1993). In some cases, these high costs may represent up to 85% of hatchery costs in bivalve culture (Coutteau & Sorgeloos 1992). In addition, production of microalgae is not reliable because of variation in food value, rapid die-offs and bacterial contamination. As a result of these problems associated with live algal production, there is a strong need for alternative diets for bivalves to reduce costs of nursery production.

Several alternative diets have been reported as partial replacements for living algal diets for Manila clams (Albentosa et al. 1989, Laing et al. 1990, Laing & Millican 1992, Curatolo et al. 1993, Coutteau et al. 1994, Albentosa et al. 2002). Recently, successful use of heterotropbically grown Schizochytrium sp. and Haematococcus pluvialis has been described in feeding experiments with clams and mussels (Boeing 1997, Langdon & Onal 1999). Boeing (1997) reported that up to 40% w/w of a mixed algal diet (Tetraselmis suecica and Chaetoceros sp.) could be replaced with heterotrophically grown Schizochytrium sp. for Manila clams without significantly reducing their growth rate. Langdon and Onal (1999) showed that juvenile mussels Mytilus galloprovincialis could be cultured on a spray-dried algal diet of 50% w/w Schizochytrium sp. and 50% w/w Spirulina sp. without significantly reducing growth rate compared with that of mussels fed on living algae. Furthermore, growth of mussels could be significantly improved by adding this spray-dried diet to a 1/4 ration of living algae.

In this study, the results of growth experiments are reported with juvenile Manila clams, Tapes philippinarum, fed combinations of a spray-dried, Schizochytrium-based product (Docosa Gold, Sanders Brine Shrimp) or a spray-dried, Haematococcus pluvialis-based product (Sanders Brine Shrimp) either alone, or as supplements for rations of living algae.

Addition of naturally occurring silt to algal diets is known to increase the growth of bivalves (Murken 1976, Kioboe et al. 1981, Ewart 1983). Therefore, the potential growth effects of additions of bentonite, a naturally occurring silt, were also investigated in this study.

MATERIAL AND METHODS

Culture Conditions

Feeding experiments were carried out with batch-cultured juvenile Manila clams, obtained from Taylor Shellfish Farms, Washington. Groups of 15 seed were placed in 10-L plastic containers. Cultures were agitated with strong aeration to keep food and clay particles in suspension. Culture water was changed every 2 to 3 days using a series of cartridge-filtered (10 [micro] 51 [micro] and 1 [micro], respectively) water at 30-35 ppt at a temperature of 19 [+ or -] 1[degrees]C. Each growth experiment lasted 3 weeks with triplicate cultures set up for each test diet.

Diets and Rations

Animals were fed twice daily on either living microalgal diets or nonliving, spray-dried diets of Schizochytrium sp. or Haematococcus pluvialis. Spray-dried diets were supplied by Sanders Brine Shrimp Company, Utah. Living algal diets were cultured under continuous light according to methods described by Breese and Malouf (1975).

Stock suspensions of spray-dried diets were prepared daily by mixing aliquots of the diets with distilled freshwater. The suspensions were sonicated for 30 sec to minimize clumping and stored at 5[degrees]C. A full dietary ration for each treatment was defined as 25 mg dry weight of diet added to each 10-L culture, resulting in a food concentration of 2.5 g [micro] dry weight [mL.sup.-1]. This concentration was equivalent, in terms of dry weight, to a mixed algal diet of 50,000 cells [mL.sup.-1] Tahitian Isochrysis galbana (T-ISO) and 50,000 cells [mL.sup.-1] Chaetoceros calcitrans (Cc), assuming a T-ISO cell weight of 2 x [10.sup.-8] mg and a Cc cell weight of 3 x [10.sup.-8] mg (Urban & Langdon 1984). A full ration of 2.5 g [micro] dry weight [mL.sup.-1] of Schizochytrium sp. resulted in a cell concentration of 25,000 cells [mL.sup.-1] (Gouthro et al. 1998). A full ration of 2.5-[micro]g dry weight [mL.sup.-1] of Haematococcus pluvialis resulted in a cell concentration of 1500 cells [mL.sup.-1] with an average individual cell size of 14 [micro] (Onal per. obs.).

In Experiment 1, Manila clams were fed twice a day on either x0 (starved), x1/4, x1/16 or x1 a full ration of a mixed algal diet consisting of T-ISO and Chaetoceros calcitrans (Cc). Each of these algal rations was either fed alone or with supplements of a full ration of spray-dried Schizochytrium sp. (SZ) or Haematococcus pluvialis (H). Full rations of live algae were increased from 50,000 cells [mL.sup.-1] (125 x [10.sup.-6] mg/L) to 70,000 cells [mL.sup.-1] (175 x [10.sup.-6] mg/L) during the experiment, equivalent to about 2% to 1.5% of the wet weight of spat, respectively, to meet increased food requirements as spat grew in size. Partial algal rations and rations of spray-dried diets were increased similarly to ensure dietary consistency throughout the experiment. Average initial individual clam live and dry organic weights were 37.5 mg and 2.9 mg, respectively.

In Experiment 2, clams were fed twice a day on either x0, x1/4 or x1 of a ration of a mixed diet consisting of 25,000 cells [mL.sup.-1] T-ISO and 25,000 cells [mL.sup.-1] Cc, equivalent to about 2.6% to 1.2% of the wet weight of spat, respectively. Each of the algal rations was either fed alone or with supplements of x0, x1/2, x1 or x2 full rations of spray-dried H. Ration were held constant (50,000 cell [mL.sup.-1]) during the experiment. In addition, calcium bentonite (B; American Colloid Ltd., Alabama) was added at 2 mg/L to some dietary treatments to test the effects on clam growth. Average initial individual clam live and organic weights were 32.4 mg and 2.1 mg, respectively.

Determination of Growth and Survival

At the beginning of each experiment, average initial live and organic weights of a group of randomly selected 15 clams from the experimental population were determined. Then, groups of 15 clams were randomly selected for each treatment from the experimental population, weighed and added to each container. At the end of each experiment, percent survival was determined and final group live and dry organic weights were compared with initial weights.

Dry organic weights were determined by first determining the dry weights of groups of clams (60[degrees]C for 48 h) followed by determination of their ash weights (450[degrees]C for 24 h). Ash weights were then subtracted from dry weights to determine dry organic group weights. Final group weights were adjusted for mortalities by assuming that if dead clams had survived to the end of the experiment then they would have grown at the same rate as living clams in their group.

Statistics

Data were analyzed by ANOVA and MANOVA using the statistical software package Statview 5 (SAS Institute Inc., USA). The suitability of the data for analysis by ANOVA was checked by viewing normal probability plots of residuals and by Bartlett test for homogeneity of variance at the 5% level of significance. Percent data were transformed when necessary to meet the assumptions of ANOVA. To determine differences among treatments, Tukey HSD Multiple Range tests were carried out (P < 0.05).

RESULTS

Effect of Supplements Added to Full or Partial Rations of Living Algae

Clam survival was 100% in all cultures. Overall, there was a significant (ANOVA; P < 0.0001) improvement in clam growth rate (% wet weight increase) when supplements of SZ or H were added to live algal rations, except when added to full algal rations (Fig. 1A). The highest percent increase in clam wet weight occurred with 1/4 ration of live algae and a supplement of H. Growth of clams fed this latter supplemented diet was not significantly different (Tukey HSD; P > 0.05) from that of clams fed on a full live algal ration alone.

[FIGURE 1 OMITTED]

The highest percent increase in clam dry organic weight occurred with a full ration of live algae plus a supplement of H (Fig. 1B). Growth of clams fed this latter supplemented diet, however, was not significantly different (Tukey HSD; P > 0.05) from that of clams fed on a full live algal ration alone. Growth (% wet and organic increase) of clams fed dietary supplements of SZ or H alone was significantly greater (Tukey HSD; P > 0.05) than that of starved clams. Starved clams lost organic weight and showed little increase in wet weight.

Effect of Calcium Bentonite (B) on Clams Fed on Living Algae With Supplements of H

Clam survival was 100% in all treatments except in one culture (H + B) with a survival of 97%. The highest percent increase in clam wet weight occurred with 1/4 of a full ration of living algae supplemented with H and additions of bentonite (Fig. 2A); however, the increase in wet weight of clams fed on this diet was not significantly (Tukey HSD; P > 0.05) greater than that of clams fed on other rations, except clams fed on 1/4 full ration alone (Tukey HSD; P < 0.0001). The highest percent increase in clam organic weight occurred with full ration of algae (Fig. 2B; Tukey HSD; P < 0.05). The organic weight of clams that received additions of bentonite was not significantly different from that of clams that did not receive bentonite (Tukey HSD; P > 0.05). Starved clams showed little increase in wet and organic weights and were significantly less than weights of clams in all the other treatments (Tukey HSD; P > 0.05).

[FIGURE 2 OMITTED]

DISCUSSION

Heterotrophically grown, spray-dried SZ and H were found to support Manila clam growth when added as supplements to rations of a mixed algal diet of T-ISO and Cc. Up to 75% reduction in the use of live algae could be achieved by addition of either SZ or H, without significantly reducing wet growth of clams. Clam growth could be improved when supplements of SZ or H (1.25 x [10.sup.-3] g/L) were added to partial rations of live algae but not when added to full algal rations. Increase in dry organic weight was significantly less when 75% of the live algae ration was substituted with either SZ or H, compared with growth of clams fed on FR alone. Discrepancies between changes in live weight and dry organic weights of bivalves in response to diets have been reported elsewhere (Lamela et al. 1996, Albentosa et al. 2002) and may indicate differences in nutritional requirements for shell versus tissue growth.

Various levels of substitution for live algal diets with dried algae have been reported previously in feeding experiments with Manila clams. For example, Curatolo et al. (1993) found that 40% of a ration of live Cc could be substituted with dried Tetraselmis suecica without a reduction in growth, compared with growth of Manila clams fed entirely on live Cc. Albentosa et al. (2002) reported 50% substitution of T-ISO with wheat-germ flour without affecting growth of Manila clam spat. Higher possible substitution levels of live algae with SZ or H may have been because of their high protein contents (39% and 25% w/w, respectively; Langdon & Onal 1999) compared with that of T. suecica (14% to 23% w/w; Laing & Milican 1992, Albentosa et al. 1996) and T-ISO (27.8%, Albentosa et al. 2002). Kreeger and Langdon (1994) showed that growth rates of juvenile Mytilus trossulus were positively correlated with dietary protein content over the range 27.8% to 42.6% w/w protein.

In contrast, increased dietary lipid levels have not been reported to improve Manila clam growth rates; for example, Caers et al. (1998) reported that supplements of the fatty acid docosahexaenoic acid (DHA; 22:6n-3) added to live algae did not improve the growth of Manila clam spat fed on T. suecica, which contained the fatty acid eicosapenthatonic acid (EPA; 20:5n-3) but only trace amounts of DHA. Laing et al. (1990) proposed that Manila clams could produce DHA (22:6n-3) by chain elongation and desaturation of precursors of shorter chain length. This argument seems to be in agreement with the results obtained in this study. Manila clam spat grew better with supplements of H compared with SZ that contained 0% and 33% w/w DHA, respectively (Langdon & Onal 1999). The high DHA content of SZ may be responsible for the good growth of mussel juveniles fed on this diet (Langdon & Onal 1999), whereas it does not seem to be beneficial for Manila clam spat. Therefore, Manila clams may not be dependent on dietary sources of DHA.

The slightly better clam growth with supplements of H compared with supplements of SZ may be because of the higher carbohydrate content of H (37% w/w) versus 18% w/w for SZ (Langdon & Onal 1999). Similarly, higher growth rates of clams fed carbohydrate rich (>50% w/w) Tetraselmis have been reported elsewhere. For example, Laing and Verdugo (1991) reported that Manila clam spat fed on a 20% ration of Cc and spray-dried T. suecica grew significantly better than spat fed on a 100% ration of live Cc alone. Laing and Millican (1992) also found that Manila clams fed on a mixture of 10% live Skeletonema costatum and 90% dried T. suecica grew at similar rates to those of clams fed on a mixed live algal diet of T. suecica and S. costatum. High growth rates of clams fed on microalgae rich in carbohydrate suggest low dietary protein requirements and may indicate carbohydrate sparing of dietary protein and lipid requirements (Albentosa et al. 1996).

Growth of Manila clams fed entirely on spray-dried SZ or H was not satisfactory. However, Langdon and Onal (1999) were able to grow mussels successfully on mixtures of dried SZ and dried Spirulina sp. without reducing mussel growth compared with that of mussels fed on full rations of live algae. It is possible that the digestive capabilities and/or dietary requirements of clams are different from those of mussels. Mussels may contain a more diverse suite of digestive enzymes that help them digest a greater variety of food types and they may be more efficient than clams in digesting dried algae. Examination of fecal strands of clams fed on SZ clearly showed some unbroken cells (Onal 2001). If the clams were not able to physically breakdown the walls of SZ, it is likely that they could not efficiently use the nutrients inside the cells. Production of spray-dried H, on the other hand, involves breakdown of the cell walls to assure a better bioavailability of the natural astaxanthin in feeds for aquaculture species. This lack of wall integrity may have resulted in increased digestibility and bio-availability of the cell contents and may explain the slightly higher growth rates of clams fed on supplements of H compared with those of clams fed on supplements of SZ.

Additions of bentonite did not have any beneficial effect on growth of Manila clams. Albentosa et al. (1997) also reported that the addition of kaolin to freeze-dried algae had no significant effect on the growth of Tapes decussates seed, in spite of a slight increase in the efficiency of absorption in the presence of kaolin compared with the same diet without kaolin. Similarly, Knauer and Southgate (1996) reported that addition of 5 or 20 mg/L kaolin to spray-dried Spongiococcum excentricum did not result in any significant improvement in the growth of Crassostrea gigas spat. In contrast, Kiorboe et al. (1981), Ewart (1983) and Ali (1983) reported positive effects of additions of clays on growth of bivalves. For example, Kiorboe et al. (1981) reported that adding 5 mg/L of natural silt to microalgae fed to Mytilus edulis juveniles improved their growth by 30% to 70%. The positive effect of clay on the growth of bivalves could be either because of increased breakdown of the ingested diet (mechanical abrasion of ingested cells) or delivery of dissolved organic matter adsorbed onto the surface of clay particles. Although clay particles may have increased nutrient availability in this study, it was not helpful in improving clam growth perhaps because of limiting essential nutrients of the spray-dried diets.

Heterotrophic production of algae and other microorganisms has several potential advantages over photosynthetic production, such as rapid growth, lower production costs and higher maximum cell densities (Barclay et al. 1994). In addition, concentrations of specific nutritional ingredients, such as long chain fatty acids and caretenoids, can be enriched in heterotrophically-grown organisms (Barclay & Zeller 1996). Up to 75% substitution levels for live algae achieved in this study coupled with lower cost of heterotrophically-grown, spray-dried algae ($25 to $73 USD per kg; Sanders Brine Shrimp) will allow more economic production of clams and may simplify hatchery-nursery procedures, leading to more consistent production of bivalve spat.

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EBRU ONAL, (1) CHRIS LANGDON (1) AND UMUR ONAL (2)

(1) Coastal Marine Experiment Station, Department of Fisheries and Wildlife, Oregon State University, Newport, Oregon 97365; (2) Canakkale Onsekiz Mart University Fisheries Faculty, Department of Aquaculture Canakkale, 17100 Turkey

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