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1 Aquaculture 279 (2008) Contents lists available at ScienceDirect Aquaculture journal homepage: Enriching rotifers with premium microalgae. Isochrysis aff. galbana clone T-ISO Martiña Ferreira, Ana Maseda, Jaime Fábregas, Ana Otero Fac. of Pharmacy, Department of Microbiology and Parasitology, Universidade de Santiago de Compostela, Santiago de Compostela, Spain ARTICLE INFO ABSTRACT Article history: Received 29 December 2007 Received in revised form 21 March 2008 Accepted 24 March 2008 Keywords: Brachionus plicatilis Microalgae Isochrysis Nutrition Semi-continuous culture Biochemical composition The effect of semi-continuous culture on the nutritional value of microalgae was tested in the rotifer Brachionus plicatilis in short-term enrichment experiments. Isochrysis aff. galbana clone T-ISO was cultured semi-continuously with renewal rates from 10 to 50% of the volume of the culture per day and used to feed the rotifers. After 24 h, dramatic differences in dry weight and protein, lipid and carbohydrate contents were observed in the rotifers depending on the renewal rate applied to the microalgal culture. Rotifers fed T-ISO cultured with low renewal rates showed low dry weight and organic content, whereas rotifers fed microalgae from nutrient-sufficient, high renewal rate cultures showed higher dry weight and increases up to 60% in protein, 35% in lipid and 100% in carbohydrate contents. Feed conversion rate (FCR) and organic FCR decreased with increasing renewal rates, indicating a more efficient assimilation of the microalgal biomass obtained at high growth rates. The fatty acid profile of rotifers reflected that of T-ISO, with maximum content of polyunsaturated fatty acids (PUFAs), n-3 fatty acids and docosahexaenoic acid (DHA) being found in the rotifers fed microalgae from the renewal rate of 40%. Results demonstrate that the biochemical composition of B. plicatilis is strongly modified through the use of semi-continuous cultures of microalgae in short-term enrichment processes. This technique provides an excellent tool to improve the nutritional value of the live feed used in fish larvae cultures Elsevier B.V. All rights reserved. 1. Introduction Microalgae play an essential role in aquaculture since they constitute the basis of the food chain, being the main diet for molluscs and initial larval stages of some crustacean and also for the production of live feed which is used for first-feeding of fish and crustacean larvae. The conditioning of water in fish larval tanks, known as green water techniques, constitutes another important use of microalgae for aquacultural purposes. However, microalgae are considered the bottleneck for the culture of molluscs (Persoone and Claus, 1980) and a serious limitation for the production of larvae of diverse marine finfish species, as their production is expensive and laborious (Wikfors and Ohno, 2001). One of the major applications of microalgae in aquaculture is the enrichment of rotifers in order to improve their nutritional content before being fed to fish larvae. Although artificially enriching diets are widely used, there is a growing trend to include microalgae in enrichment procedures. The effect of different microalgal species on the proximate biochemical composition of Brachionus plicatilis has been reported (Ben-Amotz et al., 1987; Whyte and Nagata, 1990; Whyte et al., 1994) demonstrating the importance of the biochemical composition of the food alga in modulating the biochemical composition of the filter-feeder. Good quality microalgae constitute an excellent diet for enrichment since they provide rotifers with essential fatty acids (Dhert et al., 2001), but frequently Corresponding author. Tel.: x16913; fax: address: mpaotero@usc.es (A. Otero). these high quality microalgae are not available in large enough quantities. The traditional culture systems, consisting of tanks, large cylinders or polyethylene bags that are used in batch mode often result in low productivity (Borowitzka, 1997) and do not allow control of the biochemical composition of the obtained biomass (Otero et al., 2002). Continuous and semi-continuous culture has been proposed as an alternative to the generally used batch culture methods for the production of microalgae (Borowitzka, 1997). These systems not only provide higher and constant supplies of microalgal biomass, but their controlled conditions permit manipulation of the biochemical composition of the algal cells (Sukenik and Wahnon, 1991; Otero and Fábregas, 1997; Otero et al., 1997). Hence, selecting the most appropriate conditions in continuous or semi-continuous cultures can strongly enhance the nutritional value of the microalgae. This has been demonstrated in culture experiments with filter feeders, in which an increase in the dilution rate of the microalgal culture improved assimilation efficiency in B. plicatilis (Scott, 1980), and somatic growth, survival and reproductive rate in Artemia (Fábregas et al., 1998, 2001). Although the modification of the biochemical composition of rotifers through the use of different species of microalgae is widely documented (Ben-Amotz et al., 1987; Frolov et al., 1991; Carić et al., 1993; Whyte et al., 1994), very few studies have approached the application of controlled-composition microalgae from continuous or semi-continuous cultures to improve the nutritional value of live feed (Øie et al., 1994; Fábregas et al., 1998, 2001). In this work, we propose the use of semi-continuous cultured microalgae for the short-term enrichment of B. plicatilis, studying the effect of the renewal rate applied to semi /$ see front matter 2008 Elsevier B.V. All rights reserved. doi: /j.aquaculture

2 M. Ferreira et al. / Aquaculture 279 (2008) continuous culture of the marine microalga Isochrysis aff. galbana clone T-ISO, a species commonly used in the enrichment of rotifers for fish larvae because of its good fatty acid profile, on the biochemical composition of the rotifers. 2. Materials and methods 2.1. Isochrysis aff. galbana clone T-ISO The marine microalga Isochrysis aff. galbana clone T-ISO (CCMP 1324) was cultured in 30 mm diameter glass tubes under a circadian light:dark cycle (12 h:12 h) and a light intensity of 162 μmol photon m 2 s 1 at a temperature of 21 C. Each tube contained 80 ml of culture medium, prepared with autoclaved seawater (salinity 35 ) enriched with nutrients (KNO 3,4mM;NaH 2 PO 4,0.22mM;ZnCl 2,2.29μM; MnCl 2,3.37μM; Na 2 MoO 4,2.72μM; CoCl 3,0.38μM; CuSO 4,0.465μM; ferric citrate, 21.2 μm; thiamin,1 mg l 1 ;biotin,13.4μgl 1 ; vitamin B 12,9.4μgl 1 ;EDTA, 14 μmol l 1,modified from Fábregas et al., 1985). Continuous aeration (230 ml min 1 ) was provided through glass capillaries (diameter 1 mm), supplemented intermittently with CO 2 in order to keep ph below 8. Cultures were inoculated at a cell density of cells ml 1 and once early stationary phase was reached, the semi-continuous regime was started, with daily renewal of 10, 20, 30, 40 and 50% of the culture volume. Renewals were carried out with autoclaved seawater enriched at the same nutrient concentration. Three replicates were set for each condition. Cell density was assessed daily in the harvested cultures by microscope cell counting using a Neubauer haemocytometer. Once the steady-state was achieved, the cultures were maintained for 1 week before being used for enrichment of the rotifers and for biochemical analyses B. plicatilis B. plicatilis were cultured routinely in soft-aerated 6 l flasks with autoclaved seawater (salinity 35 ) and fed a mixture of Isochrysis galbana T-ISO, Tetraselmis suecica, Nannochloropsis gaditana and Rhodomonas lens. Periodically, rotifers were filtered with a 45-μm mesh size nylon net, rinsed with distilled water and transferred to a new flask containing autoclaved seawater. Previous to the experiment, rotifers were filtered and deprived of food for 12 h in order to avoid interferences from the gut content on the results. To carry out the enrichment, rotifers were transferred to flasks containing 700 ml of autoclaved seawater, at a final density of 200 individuals ml 1. The food ration was calculated on a cell number basis (27,000 cells of T-ISO per individual equivalent to a cell density of cells ml 1 ). This ration was sufficient to feed the rotifers during 24 h, as evidenced by a slight residue of microalgal cells in the rotifer cultures after that period. Patiño (1995) concluded that a ration of 2000 cells of T. suecica per rotifer, equivalent to 400 ng of dry weight, was enough to sustain feeding and provided a constant ingestion rate avoiding the existence of periods of gut repletion followed by intervals of decreased filtration activity. An average dry weight of 18 pg T- ISO cell 1 regardless of the culture conditions was assumed for the calculation of the food ration, even thought the increase of renewal rate causes a decrease in the volume and organic content of microalgal cells (Fábregas et al., 1996). Three replicates of rotifer cultures were set for each renewal rate of the microalga. After 24 h of feeding, rotifers were collected. A sample of rotifers was collected after the 12 h starvation period and before the enrichment as a control group Sampling procedures For dry weight determination, samples of 5 ml of microalgal culture or 100 ml of rotifer culture were filtered on previously weighed precombusted Whatman GF/C fiberglass filters. Microalgal samples were washed three times with 5 ml of 0.5 M ammonium formate in order to remove salts and rotifer samples were rinsed with distilled water. Filters were dried overnight at 80 C and dry weight determined gravimetrically. For Fig. 1. Cell density (bars) and productivity ( ) of semi-continuous cultures of Isochrysis aff. galbana clone T-ISO. biochemical analyses, microalgal samples (5 to 10 ml) were collected by centrifugation and immediately frozen at 20 C. 80 ml of rotifer culture were collected on a sieve (45 μm mesh size), rinsed with distilled water and frozen at 20 C for fatty acid analysis. The remaining rotifers were also collected on a sieve and freeze dried for the rest of the biochemical analyses. Feed conversion rates (FCR) were calculated as the ratio between dry or organic (protein+lipid+carbohydrate) weight of T-ISO supplied and the equivalent increase in rotifer biomass Biochemical analyses Protein content was measured by the Lowry et al. (1951) method as modified by Herbert et al. (1971), and carbohydrates were measured by the phenol-sulfuric acid method (Kochert, 1978). Lipids were extracted by the method of Bligh and Dyer (1959) and measured by the charring method (Marsh and Weinstein, 1966). For C H N determination, freeze dried microalgal samples were analyzed with an autoanalyzer (Carlo Erba EA 1108, Rodano, Italy). For fatty acid analysis, lipids were extracted and subjected to methanolysis with 5% HCl in methanol at 85 C (Sato and Murata, 1988), extracted with hexane and analyzed with a gas chromatogram mass spectrometer (MD-800, Fisons Instruments, Beverly, Mass.), using triheptadecanoin as the internal standard (Sigma, St. Louis, Mo). All the analyses were carried out in triplicate Statistical analysis The statistical treatment of the data was performed with the nonparametrical Mann Whitney U test. All analyses were conducted using SPSS 14.0 for Windows. 3. Results Cell density in steady-state T-ISO cultures decreased as renewal rates increased, whereas productivity showed a parabolic-shaped development, reaching its maximal value with the renewal rate of 30% (Fig. 1). The different renewal rates modified nutrient and light availability, thus affecting the biochemical composition of the algal cells. The C:N ratio decreased to one half between the renewal rates of 10 and 40% (Fig. 2) and remained stable with higher renewal rates (Fig. 2). The stability in the C:N ratio indicates nitrogen saturation in the microalgal cultures (Otero et al., 1998). Higher nitrogen availability allowed microalgal cells to accumulate protein contents of 5.01 and 4.16 pg cell 1 at the renewal rates of 40 and 50% respectively, while only 3.41 pg cell 1 of protein was obtained with a renewal rate of 10%. The Mann Whitney U test showed that these protein contents were significantly higher (Pb0.05) than those obtained in nitrogen-limited conditions, i.e. renewal rates from 10

3 128 M. Ferreira et al. / Aquaculture 279 (2008) Fig. 2. Composition of the organic fraction of Isochrysis aff. galbana clone T-ISO cultured semi-continuously with different renewal rates (left) and Brachionus plicatilis enriched for 24 h with the same microalgal cultures (right), expressed as pg cell 1 and ng rotifer 1 respectively. Crosshatched bars, protein; hatched bars, lipid; open bars, carbohydrate;,c: N ratio. C: rotifer control sample, harvested before the start of the enrichment. to 30% (Fig. 2). Culture conditions also modified carbohydrate and lipid contents. Carbohydrate was the fraction most affected by the increase of renewal rates, with cell content decreasing significantly (Pb0.05) from pg cell 1 to 2.69 pg cell 1 between the renewal rates of 10 and 40%, whereas lipids decreased significantly from 6.47 pg cell 1 at the renewal rate of 10% to 4.23 pg cell 1 at the renewal rate of 30% (Pb0.05) and stabilized onwards. As a consequence, the total organic content of T-ISO cells decreased as renewal rate increased (Fig. 2). Variations in the biochemical composition of T-ISO caused by the different renewal rates strongly modified its nutritional value for B. plicatilis. Final rotifer dry weight was inversely proportional to the weight of microalgae supplied, since an equal number of cells per individual were provided but cell dry weight decreased with increasing renewal rates (Fig. 2). Consequently, feed conversion rate (FCR), calculated as the ratio between dry weight of T-ISO supplied and the increase in rotifer biomass, underwent a 2-fold decrease as nutrient availability in the microalgal culture medium became higher and reached its lowest value in nitrogen-saturation conditions, i.e. with renewal rates of 40 and 50%, indicating that rotifers assimilated more efficiently microalgae from the higher renewal rates. Organic FCR, calculated as the ratio between organic weight of T-ISO supplied and the increase in organic biomass or rotifers, showed the same trend, but its decrease was even steeper, more than 3-fold between the renewal rates of 10 and 50% (Table 1). Control rotifers, sampled just before starting the enrichment experiment, showed low dry weight, 115.3±16.3 ng individual 1 (Fig. 2). After the enrichment, rotifers showed significant (Pb0.05) increases in dry and organic weight with regard to the control individuals. The groups of rotifers enriched with T-ISO from renewal rates from 10 to 30% reached similar dry weights, around 300 ng individual 1, whereas in rotifers fed microalgae from the higher renewal rates, the dry weight continued to increase, reaching 460.7±11.2 ng individual 1 at a renewal rate of 50%. Dry weight was significantly different in all groups (Pb0.05), despite the similar values observed in rotifers enriched with T-ISO from nutrient-limited renewal rates, i.e. 10 to 30% (Table 1). Organic content followed the same pattern as dry weight and remained almost constant in rotifers enriched with T-ISO cells from nutrient-deficient cultures, but increased markedly in rotifers fed nutrient-sufficient microalgae. This development contrasts with the drop in the organic content of T-ISO as renewal rate increased. No statistical differences were found between the protein, lipid or carbohydrate content of the rotifers enriched with microalgae from renewal rates of 10, 20 and 30%. In contrast, for rotifers fed T-ISO cells from nitrogen-sufficient cultures, the same three components, protein, lipid and carbohydrate, increased significantly with regard to nutrient-limited conditions, and significant differences between renewal rates of 40 and 50% were also observed (Pb0.05). Protein content varied between 91 and 100 ng individual 1 in rotifers fed microalgae from the renewal rates from 10 to 30% and increased to 158 ng individual 1 in rotifers fed cells from the renewal rate of 50%. The increase in lipid content was less, from 33 ng individual 1 at the renewal rate of 10% to 45 ng individual 1 at the renewal rate of 50%, while carbohydrate content almost doubled, from 28 to 49 ng individual 1 (Fig. 2). Despite the differences found in the gross biochemical composition expressed as individual content, percentages of protein, lipid and carbohydrate in the organic fraction of the rotifers did not change significantly, being 61 63% for protein, 17 21% for lipid and 17 20% for carbohydrate. Control rotifers showed a higher percentage of protein, 69.5%, and a lower percentage of carbohydrate, 11%, compared to enriched rotifers (data not shown). In agreement with the trend observed for FCR and organic FCR, percentages of recovery of the organic fractions tended to increase with the renewal rate. Percentages of protein recovery did not follow any particular trend and ranged from 58 to 67% between the renewal rates of 10 and 40%; however, the highest value, 95%, was obtained with the renewal rate of 50%. Lipid and carbohydrate recoveries underwent a continuous increase with renewal rate, from 13 to 27% for lipid and from 7 to 55% for carbohydrate (Table 1). Culture conditions affected the fatty acid profile of the microalga and those changes were reflected in the rotifer. The increase in the renewal Table 1 Food supply and conversion efficiencies of Brachionus plicatilis enriched for 24 h with T-ISO from semi-continuous cultures Renewal rate 10% 20% 30% 40% 50% T-ISO consumed (µg ml 1 ) Organic weight of T-ISO consumed (μg ml 1 ) Rotifer dry weight (ng individual -1 ) a ± b ± c ± d ± e ± FCR (weight of T-ISO consumed/increase in rotifer weight) Organic FCR (organic weight of T-ISO consumed/increase in rotifer organic weight) % Protein recovery % Lipid recovery % Carbohydrate recovery Values in the same row with different superscripts are significantly different (Pb0.05).

4 M. Ferreira et al. / Aquaculture 279 (2008) Table 2 Fatty acid composition (as percentage of total fatty acids) of Isochrysis aff. galbana clone T-ISO cultured in semi-continuous regime and Brachionus plicatilis enriched with those microalgae and total fatty acid content, expressed as pg T-ISO cell 1 or as ng individual 1 Renewal rate 10% 20% 30% 40% 50% T-ISO Rotifer T-ISO Rotifer T-ISO Rotifer T-ISO Rotifer T-ISO Rotifer 14: ± ± ± ± ± ± ± ± ± ± : ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± :2n ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± :4n ± ± ± ± ± ± ± ± ± ± :4n ± ± ± ± ± ± ± ± ± ± :5n ± ± ± ± ± ± ± ± ± ± :6n ± ± ± ± ± ± ± ± ± ±0.28 Others ± ± ± ± ± ± ± ± ± ±3.11 Total saturated 39.28± ± ± ± ± ± ± ± ± ±2.65 Total monounsaturated 29.73± ± ± ± ± ± ± ± ± ±1.76 Total polyunsaturated 29.50± ± ± ± ± ± ± ± ± ±2.93 n ± ± ± ± ± ± ± ± ± ±2.48 n ± ± ± ± ± ± ± ± ± ±0.73 n-3 /n Total fatty acids 1.37± ± ± ± ± ± ± ± ± ± Includes fatty acids with abundances b4%: 12:0, 16:1n-9, 16:2n-6, 16:2n-4, 18:0, 18:1n-7, 18:3n-6, 20:0, 20:1n-9, 20:2n-6, 20:3n-6, 22:0, 22:1n-9 and 22:5n-6. rate resulted in a strong increase in the percentage of polyunsaturated fatty acids (PUFAs) in T-ISO, from 29.50% of the total fatty acids with a renewal rate of 10% to 46.36% in the cultures with a renewal rate of 40% (Table 2). The main polyunsaturated fraction corresponded to n-3 fatty acids, which also reached its maximum percentage, nearly 39%, with a renewal rate of 40%. The most abundant PUFA in T-ISO was 18:4n-3, that increased from 11.96% at the renewal rate of 10% to 20.48% at the renewal rate of 40%. 22:6n-3 (DHA) was the second most abundant PUFA in T-ISO andfollowedthesametrendas18:4n-3,increasingfrom6.28to9.88% between the renewal rates of 10 and 40%. Eicosapentaenoic acid (20:5n- 3, EPA) content was very low in T-ISO, ranging between 0.41 and 0.53% of the total fatty acids and not showing any particular trend (Table 2). The modifications of the fatty acid composition of B. plicatilis followed the same pattern of the alga, but they were less marked. The PUFA fraction increased only from 30 to 36% whereas the maximum proportion of n-3 fatty acids, achieved in the rotifers enriched with T-ISO from the renewal rate of 40%, was 26.5%. The percentage of DHA increased slightly with increasing renewal rates, up to 5% in the rotifers fed microalgae from the renewal rate of 40%. The percentage of EPA also reached its maximum value, 1.72%, in the renewal rate of 40% (Table 2). At the renewal rate of 50% the percentage of PUFAs decreased slightly in both T-ISO and B. plicatilis (Table 2). 4. Discussion The effect of the composition of a microalgal diet on the proximate biochemical composition of B. plicatilis can be readily observed when culture experiments are carried out for several days. Whyte et al. (1994) found significant differences on the composition of rotifers cultured for at least 3 weeks with different microalgal species, whereas Ben-Amotz et al. (1987) found a close relation between both the gross biochemical composition and lipid profile of rotifers and those of their algal diet. Strong variations in sugar composition could be detected in B. plicatilis after 15 days of feeding with different species of algae (Whyte and Nagata, 1990). However no differences were observed in the composition of rotifers fed different microalgal species when the algae were cultured under the same conditions of semi-continuous regime (Øie et al., 1994). On the contrary, the results of experiments on short-term enrichment are more diverse. Although Scott and Baynes (1978) attributed only minor effects to the species of microalga used on the biochemical composition of rotifers sampled at 24 h intervals, other studies found changes in the composition of the rotifers after 6 h of enrichment, and the existence of correlation between the composition of the rotifers and that of the microalgae after feeding periods of 48 h (Frolov et al., 1991). Continuous increases in the individual protein and lipid contents and the dry weight of B. plicatilis enriched with microalgae up to 48 h have also been reported (Reitan et al., 1997). A change in the rotifer's diet from yeast and emulsified oil to T-ISO modified the percentages of some fatty acids after a feeding period of 23 h (Lie et al., 1997). After 24 h of enrichment with T-ISO cultured in semi-continuous regime, rotifer dry and organic weight underwent up to 4-fold and 5-fold increases respectively, with increasing renewal rate. Droop and Scott (1978) calculated that the gut content from actively feeding rotifers constituted approximately 12% of the total rotifer carbon (Scott, 1980). The incorporation of nutrients into animal tissue is expected to be low in short-term feeding experiments, and modifications of the biochemical composition have been attributed mainly to the presence of feed in the digestive tract, whose contribution to the organism total weight, as seen above, is also limited. In contrast, our results exceed the figure proposed by Droop and Scott (1978) for gut content and hence suggest a rapid and high assimilation of microalgal nutrients into the rotifer body tissue, that strongly depends on the culture conditions of the microalgae. Consequently, even though it is generally accepted that rotifers act as carriers, transferring the content of their guts to fish and crustacean larvae which can easily assimilate the nutrients contained in the partially digested algal biomass (Lubzens et al., 1989; Wikfors and Ohno, 2001), changes generated in the weight and biochemical composition of the rotifer in only 24 h indicate that this might not be totally accurate. Changes in the biochemical composition of the rotifers showed two different trends. Individuals enriched with T-ISO cultured under nitrogen limitation incorporated similar contents of protein, lipid and carbohydrateregardlessoftherenewalrateappliedtothemicroalgalcultures and thus independently of both the degree of nutrient limitation and the variations in the composition of T-ISO cells. The nutritional status of these T-ISO cultures is similar to those of batch culture systems commonly used in aquaculture facilities, which are harvested at the end of the exponential phase after nutrient depletion. Once nitrogen sufficiency was reached, incorporation of nutrients from the microalga to the rotifers increased with increasing renewal rates. On the contrary, differences in nutrient availability in continuous cultures of T. suecica did not cause modifications of the biochemical composition or the survival of tiger prawn Penaeus semisulcatus larvae (D'Souza and Kelly, 2000), although the nutritional status of their microalgal cultures was not clearly established. The decrease in the content of storage compounds, carbohydrate and lipid, with high renewal rates probably enhanced the digestibility of T-ISO cells, as algae with low C:N ratios and thus high relative content of assimilable nitrogen improved assimilation efficiencies and somatic

5 130 M. Ferreira et al. / Aquaculture 279 (2008) growth in Artemia (Sick, 1976). However, neither the changes in the biochemical composition of B. plicatilis nor the evolution of FCR are directly related to the increase in renewal rate, the change of the nutritional status of the algal cultures or the evolution of the biochemical components of T-ISO cells. Similarly, Fábregas et al. (1998, 2001) could not find a single biochemical parameter of Phaeodactylum tricornutum or T. suecica cultured in a semi-continuous system with different renewal rates to explain the effect on growth and survival of Artemia. Protein and lipid contents underwent between 3-fold and 4.5-fold increases in relation to the values of initial control rotifers, higher than the data reported by Reitan et al. (1997) after 24-h enrichment with T. suecica and I. galbana, with increases between 50 and 70% with regard to the initial values. Particularly, the enrichment with T-ISO from the renewal rate of 50% resulted in individual protein content comparable to those obtained by enriching rotifers with a high-protein artificial diet (Øie et al., 1997). Experiments in turbot larvae rearing have indicated the importance of the protein content of live feed, as higher growth and survival were observed in larvae fed rotifers rich in protein (Øie et al., 1997). However, lipid emulsions proved to be more efficient than T-ISO to improve the lipid content of rotifers, as up to 100 ng lipid/ rotifer was achieved after 24 h of enrichment (Øie and Olsen, 1997; Øie et al., 1997). The increase in DHA content of T-ISO with increasing renewal rate was reflected in the rotifers. The maximum percentage of DHA observed in the rotifers (5.06%) was slightly higher than the values obtained by Takeyama et al. (1996). Although these authors did not find any difference in the content of DHA of rotifers either cultured for a week or enriched for 24 h with T-ISO, our results showed a clear effect of the DHA content of the microalga in rotifers after a 24-h period. Percentages of DHA and total n-3 fatty acids similar to those reported in our work resulted in good growth performance of gilthead sea bream larvae fed rotifers (Mourente et al., 1993). Our results confirm that using the appropriate culture techniques significantly improves the nutritional value of microalgae for the production of live feed for aquaculture purposes. Previous studies had shown the effect of microalgal semi-continuous cultures on the growth, reproductive performance and biochemical composition of filter feeders in long-term experiments (Fábregas et al., 1998, 2001), but the present work demonstrates that a significant influence of semi-continuously cultured microalgae in both the gross biochemical composition and fatty acid profile of B. plicatilis can be observed after a feeding period as short as 24 h. Since the improvement of microalgal production in aquaculture facilities is still an unresolved issue, our results are clearly encouraging toward the optimization of culture methods to produce microalgae of high nutritional value in order to reinforce the base of the aquaculture food chains. Acknowledgments We thank Ian Ratcliffe for revising the manuscript. Martiña Ferreira was supported by a FPU fellowship from the Spanish Ministry of Education and Science (ref. no. AP ). References Ben-Amotz, A., Fishler, R., Schneller, A., Chemical composition of dietary species of marine unicellular algae and rotifers with emphasis on fatty acids. Mar. Biol. 95, Bligh, E.G., Dyer, W.J., A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, Borowitzka, M.A., Microalgae for aquaculture: opportunities and constraints. J. Appl. Phycol. 9, Carić, M., Sanko-Njire, J., Skaramuca, B., Dietary effects of different feeds on the biochemical composition of the rotifer (Brachionus plicatilis Müller). Aquaculture 110, Dhert, P., Rombaut, G., Suantika, G., Advancement of rotifer culture and manipulation techniques in Europe. Aquaculture 200, Droop, M.R., Scott, J.M., Steady-state energetic of a planktonic hervibore. J. Mar. Biol. Assoc. U. K. 58, D'Souza, F.M.L., Kelly, G.J., Effects of a diet of a nitrogen-limited alga (Tetraselmis suecica) on growth, survival and biochemical composition of tiger prawn (Penaeus semisulcatus) larvae. Aquaculture 181, Fábregas, J., Herrero, C., Cabezas, B., Abalde, J., Mass culture and biochemical variability of the marine microalga Tetraselmis suecica Kylin (Butch) with high nutrient concentrations. Aquaculture 49, Fábregas, J., Cid, A., Morales, E., Cordero, B., Otero, A., Discrepancies between cell volume and organic content in semi-continuous cultures of a marine microalga. Lett. Appl. Microbiol. 22, Fábregas, J., Otero, A., Morales, E.D., Arredondo-Vega, B.O., Patiño, M., Modification of the nutritive value of Phaeodactylum tricornutum for Artemia sp. in semicontinuous cultures. Aquaculture 169, Fábregas, J., Otero, A., Dominguez, A., Patiño, M., Growth rate of the microalga Tetraselmis suecica changes the biochemical composition of Artemia species. Mar. Biotech. 3, Frolov, A.V., Pankov, S.L., Geradze, K.N., Pankova, S.A., Spektorova, L.V., Influence of the biochemical composition of food on the biochemical composition of the rotifer Brachionus plicatilis. Aquaculture 97, Herbert, D., Phipps, P.J., Stranoe, R.E., Chemical analysis of microbial cells. In: Norris, J.R., Ribbons, D.W. (Eds.), Methods in Microbiology. Academic Press, London, pp Kochert, G., Carbohydrate determination by the phenol-sulfuric acid method. In: Hellebust, J.A., Craigie, J.S. (Eds.), Handbook of Phycological Methods. Physiological and Biochemical Methods. Cambridge University Press, London, pp Lie, Ø., Haaland, H., Hemre, G.I., Maage, A., Lied, E., Rosenlund, G., Sandnes, K., Olsen, Y., Nutritional composition of rotifers following a change in diet from yeast and emulsified oil to microalgae. Aquacult. Int. 5, Lowry, O.H., Rosebrough, H.J., Farr, A.L., Randall, R.J., Protein measurement with the folin-phenol reagent. J. Biol. Chem. 193, Lubzens, E., Tandler, A., Minkoff, G., Rotifers as food in aquaculture. Hydrobiologia 186/187, Marsh, J.B., Weinstein, D.B., Simple charring method for determination of lipids. J.LipidRes.7, Mourente, G., Rodríguez, A., Tocher, D.R., Sargent, J.R., Effects of dietary docosahexaenoic acid (DHA; 22:6n-3) on lipid and fatty acid compositions and growthingiltheadsea bream (Sparus aurata L.) larvaeduringfirst feeding. Aquaculture 112, Øie, G., Olsen, Y., Protein and lipid content of the rotifer Brachionus plicatilis during variable growth and feeding condition. Hydrobiologia 358, Øie, G., Reitan, K.I., Olsen, Y., Comparison of rotifer culture quality with yeast plus oil and algal-based cultivation diets. Aquacult. Int. 2, Øie, G., Makridis, P., Reitan, K.I., Olsen, Y., Protein and carbon utilization of rotifers (Brachionus plicatilis) in first feeding of turbot larvae (Scophthalmus maximus L.). Aquaculture 153, Otero, A., Fábregas, F., Changes in the nutrient composition of Tetraselmis suecica cultured semicontinuously with different nutrient concentrations and renewal rates. Aquaculture 159, Otero, A., García, D., Morales, E.D., Arán, J., Fábregas, J., Manipulation of the biochemical composition of the eicosapentaenoic acid-rich microalga Isochrysis galbana in semicontinuous cultures. Biotech. Appl. Biochem. 26, Otero, A., Domínguez, A., Lamela, T., García, D., Fábregas, J., Steady states of semicontinuous cultures of a marine diatom: effect of saturating nutrient concentrations. J. Exp. Mar. Biol. Ecol. 227, Otero, A., Patiño, M., Domínguez, A., Fábregas, J., Tailoring the nutritional composition of microalgae for aquaculture purposes the use of semicontinuous culture techniques. World Aquac./ Aquac. Eur. 33, Patiño, M., Nutrición de Brachionus plicatilis y Artemia sp. con microalgas marinas. Ph. D. Thesis, University of Santiago de Compostela. Persoone, G., Claus, C., Mass culture of algae: a bottleneck in the nursery culturing of molluscs. In: Shelef, G., Soeder, C.J. (Eds.), Algae Biomass. Elsevier/North-Holland. Biomedical Press, Amsterdam, pp Reitan, K.I., Rainuzzo, J.R., Øie, G., Olsen, Y., A review of the nutritional effects of algae in marine fish larvae. Aquaculture 155, Sato, N., Murata, N., Membrane lipids. In: Parker, L., Glazer, A. (Eds.), Cyanobacteria, Methods in Enzymology, vol California Academic Press, San Diego, pp Scott, A.P., Effect of growth rate of the food alga on the growth/ingestion efficiency of a marine herbivore. J. Mar. Biol. Assoc. U.K. 60, Scott, A.P., Baynes, S.M., Effect of algal diet and temperature on the biochemical composition of the rotifer, Brachionus plicatilis. Aquaculture 14, Sick, L.V., Nutritional effect of five species of marine algae on the growth, development and survival of the brine shrimp Artemia salina. Mar. Biol. 35, Sukenik, A., Wahnon, R., Biochemical quality of marine unicellular algae with special emphasis on lipid composition. I. Isochrysis galbana. Aquaculture 97, Takeyama, H., Iwamoto, K., Hata, S., Takano, H., Matsunaga, T., DHA enrichment of rotifers: a simple two-step culture using the unicellular algae Chlorella regularis and Isochrysis galbana. J. Mar. Biotech. 3, Whyte, J.N.C., Nagata, W.D., Carbohydrate and fatty acid composition of the rotifer, Brachionus plicatilis, fed monospecific diets of yeast or phytoplankton. Aquaculture 89, Whyte, N.J.C., Clarke, W.C., Ginther, N.G., Jensen, J.O.T., Townsend, L.D., Influence of composition of Brachionus plicatilis and Artemia on growth of larval sablefish (Anoplopoma fimbria Pallas). Aquaculture 119, Wikfors, G.H., Ohno, M., Impact of algal research on aquaculture. J. Phycol. 37,