NAOSITE: Nagasaki University's Academic Output SITE Title Application of the minute monogonont rotifer proales similis de Beauchamp in larval rearing of seven-band grouper epinephelus septemfasciatus Author(s) Wullur, Stenly; Sakakura, Yoshitaka; Hagiwara, Atsushi Citation Aquaculture, 315(3-4), pp.355-360; 2011 Issue Date 2011-05 URL http://hdl.handle.net/10069/25161 Right Copyright © 2011 Elsevier B.V. All rights reserved. This document is downloaded at: 2016-09-14T16:48:49Z http://naosite.lb.nagasaki-u.ac.jp 1 Application of the minute monogonont rotifer Proales similis de Beauchamp in larval rearing of seven-band grouper Epinephelus septemfasciatus Stenly Wullur a,b, Yoshitaka Sakakura c, Atsushi Hagiwara a, a Graduate School of Science and Technology, Nagasaki University, Nagasaki 852- 8521, Japan b Faculty of Fisheries and Marine Science, Sam Ratulangi University, Manado 95-115, Indonesia c Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan *Corresponding author Atsushi Hagiwara Tel/fax: +81 95 819 2830 E-mail address: hagiwara@nagasaki-u.ac.jp (A. Hagiwara) 2 Abstract In comparison to the rotifer Brachionus rotundiformis, the euryhaline rotifer Proales similis has a much smaller body size (83 m in length and 40 m in width), and it may be applicable as live food for rearing marine fish larvae with a very small mouth size. A mass culture technique of P. similis was recently established, and it has already been confirmed that marine fish larvae could ingest P. similis. In the present study, we further investigated the use of P. similis as an initial food by observing larval ingestion and digestion and analyzing the nutritional profile, as well as through a 10-day larval rearing trial to investigate survival and growth. Seven-band grouper Epinephelus septemfasciatus larvae showed higher selectivity against P. similis than B. rotundiformis 4 days after hatching. The larvae digested and utilized P. similis as an energy resource as they grew, and survived until the end of the experiment. The fatty acid profile of P. similis changed according to the type of microalgae; Nannochloropsis oculata NIES2146 strain and “super fresh” Chlorella vulgaris V-12® (Chlorella Industry, Fukuoka, Japan) were used as food sources. Higher growth and survival during the initial 10 days were observed when P. similis and B. rotundiformis were co-fed to the seven-band grouper larvae. Keywords: Rotifer, Proales similis, Brachionus rotundiformis, live food, fatty acid, Epinephelus septemfasciatus. 3 1. Introduction The euryhaline rotifer Brachionus plicatilis has been used as an excellent initial live food for rearing marine fish larvae (Ito, 1960; Lubzens, 1987; Lubzens et al., 1989; Hagiwara et al., 2001, 2007). B. plicatilis is a complex of sibling species with a body length ranging between 90 and 400 m. Among these species, B. rotundiformis (Segers, 1995; Ciros-Perez et al., 2001; Gomez et al., 2002; Kotani et al., 2005; Fontaneto et al., 2007) is the smallest in size and is commonly referred to as a “super-small” (SS) -type by culturists. B. rotundiformis has been used for feeding fish larvae with small mouths, such as groupers (Okumura, 1997; Soyano et al., 2008). It is reported that grouper larvae at the initial stage (Epinephelus akaara, E. septemfasciatus, E. bruneus, Plectropomus leopardus) ingest smaller (or younger) B. rotundiformis, which contain less nutrition because they hatch in larval rearing tanks and feed on background microalgae added to the tanks at a low density (Okumura, 1997). Furthermore, there are tropical marine fish species such as Napoleon wrasse (Cheilinus undulatus) and angelfishes (family Pomacanthidae), whose larvae cannot ingest B. rotundiformis because of their extremely small mouth size (Tucker, 1998), and they require even smaller live food in the range 40-80 m at the initial feeding stages (Slamet and Hutapea, 2004; Olivotto, et al., 2006). Many wrasses (family Labridae) including C. undulatus are economically important because of their high market prices, especially in Asian countries. Marine angelfish are commercially valuable as ornamental fish. 4 However, mass larval rearing techniques have not been established for these groups, partly due to the lack of appropriate live feed for their initial feeding. Recognizing these demands for smaller-sized live feed, we have developed Proales similis as a starter live food organism for the larviculture of small mouth fishes (Wullur et al., 2009). The average body size of P. similis (83 m in length and 40 m in width) is 38% smaller and 60% narrower than the so-called SS-type Indonesian strain B. rotundiformis (Hagiwara et al., 1995; Wullur et al., 2009). P. similis can be masscultured in the same manner as euryhaline Brachionus (Wullur et al., 2009). We also confirmed that marine fish larvae, namely the seven-band grouper Epinephelus septemfasciatus larvae, could ingest P. similis (Wullur et al., 2009). In the present study, we further investigated the use of P. similis as an initial food by monitoring the ingestion, digestion, survival and growth of seven-band grouper to determine whether the feeding of P. similis is effective to enhance the survival and growth of the larvae, although larval rearing in this species is conducted by feeding socalled S or SS-type rotifers (Hagiwara et al., 2007; Soyano et al., 2008). We also conducted an analysis of the fatty acid profile of P. similis to examine whether P. similis can be nutritionally manipulated as well as whether larvae can utilize P. similis as a nutritional source so that larvae show appropriate survival and growth. 5 2. Materials and Methods 2.1. Rotifer culture The P. similis used in this study was originally collected from an estuary in Ishigaki Island, Okinawa, Japan, in July 2004 (Wullur et al., 2009). P. similis was mass-cultured in 50 l polycarbonate tanks. The water temperature and salinity were 25 o C and 25 ppt, respectively. Concentrated microalgae N. oculata NIES-2146 strain and “super fresh” Chlorella vulgaris V-12® (reviewed by Hagiwara et al., 2001), purchased from Chlorella Industry Fukuoka, Japan, were fed to the rotifers. Mass culture and nutritional enrichment were performed at the same time with these microalgae, and harvested rotifers were directly fed to fish larvae. The two microalgal species were supplied to rotifers twice a day to maintain the density at 28.8 g dry weight/ml, which corresponds to 12.5x106 and 4.3x106 cells/ml for N. oculata and C. vulgaris V-12®, respectively. These food levels are optimal for the mass culture of P. similis (Wullur et al., 2009). Rotifer population density was monitored once daily by counting the number of rotifers in triplicate 1-ml samples taken from each culture tank. During the larval rearing experiment, P. similis was raised (in 30-50 l) using the semi-continuous culture method (reviewed by Hoff and Snell, 1987; Lubzens, 1987) by replacing 1/3 of the culture water. The size distribution of each rotifer species was measured using a digital microscope (Keyence VH-8000, Keyence Corp.) at a magnification of 450x, while dry weight was determined as follows: each rotifer was debris-free siphoned from each 6 culture and filtered using a 10-m mesh plankton net to remove microalgae. The filtered rotifers were transferred to a beaker containing 250 ml diluted seawater (25 ppt) in three replications, and aliquot samples were counted to estimate the total number of each rotifer species. Further, the rotifers were re-filtered using previously weighed precombusted Whatman GF/C fiberglass filters and rinsed with distilled water to remove salt. The filters were dried overnight at 80 oC, and dry weight was determined gravimetrically using a microbalance (Mettler Toledo UMX2, Mettler-Toledo, Columbus, Ohio, USA). 2.2. Larval rearing of seven-band grouper larvae Feeding trials of seven-band grouper E. septemfasciatus larvae were conducted in 9 transparent 100-l polycarbonate cylindrical tanks following the method described by Ruttanapornvareesakul et al. (2007). The tanks were placed in the laboratory, where the temperature (24-26 oC) and photoperiod (12L:12D) were constant. Artificial seawater at a salinity of 32-34 ppt (Marine Art Hi, Tomita Pharmaceutical, Japan) was used to fill the tanks. The rearing water was not exchanged throughout the experiment. Ceramic sand (MS-0, Norra Co. Ltd., Kyoto, Japan) was placed on the bottom of each tank to help stabilize the water quality. Aeration at a rate of 50 ml/min controlled by a flow meter (Kofloc RK-1350V) was provided through an air stone placed at the center of the bottom of each tank. 7 The rearing experiment was conducted using artificially fertilized eggs of E. septemfasciatus obtained from Nagasaki Prefectural Fisheries Experimental Station on May 24, 2007. Each tank was stocked with 1,500 eggs. Oil was applied at 0.2 ml/m2 to form a film on the surface of each rearing tank to prevent surface death of the larvae (Yamaoka et al., 2000). At the onset of feeding at 4 days after hatching (DAH), rotifers were introduced in three treatments, each with three replicates. The three feeding treatments were 20 ind./ml P. similis, 20 ind./ml B. rotundiformis and a mixture of both rotifer species (each at 10 ind./ml), respectively. Rotifers were first enriched with “super fresh” C. vulgaris V-12® for around 1 week prior to the feeding of the larvae. After the addition of the rotifers, “super fresh” C. vulgaris V-12® at 5x105cell/ml was introduced to the larval rearing tanks for green water culture. The densities of the rotifers as well as “super fresh” C. vulgaris V-12® inside the larval rearing tanks were replenished daily to maintain experimental levels. Seven-band grouper larvae were reared for 10 days after hatching. At 4, 5, 6, 8 and 10 DAH, 10 larvae were sampled from each tank between the hours of 1600 and 1700, anesthetized with 0.01% MS 222 (Tricaine; Sigma Chemical Co., St. Louis, MO, USA) and fixed with 5 % formaldehyde. The mouth size (upper jaw length times 2 0.5, Shirota, 1970) and standard (notochord) length of the larvae were measured using a digital microscope (VH-6300, Keyence Corp., Japan) at a magnification of 100-175 x. Feeding incidence (percentage of larvae with rotifers in the gut) and food quantity 8 (number of rotifers in the gut) were measured by dissecting the larval gut under a stereomicroscope. Food quantity was determined by counting the number of undigested rotifer bodies as well as the trophi of digested rotifers (Akazawa et al., 2008). Trophi are the calcified jaws located at the beginning of the digestive tracts of rotifers (Kleinow et al., 1990; Kleinow, 1998; Sorensen, 2002), and they remain undigested after the other relatively soft parts of the rotifer have been digested, thus providing a more accurate estimation of food quantity. The survival rate of the larvae in all treatments was estimated by counting the number of surviving larvae at the end of the experiment. 2.3. Biochemical analysis 2.3.1 Tryptic enzyme analysis for seven-band grouper larvae The tryptic enzyme activity of seven-band grouper larvae was measured using a methods that followed that of Ruttanapornvareesakul et al. (2010) with slight modifications of Ueberschar (1995) and Araujo et al., (2001). Five larvae were sampled early in the morning prior to feeding at 5, 8 and 10 DAH, anesthetized with 0.01% MS 222 (Tricaine; Sigma Chemical Co., St Louis, MO, USA) and then frozen at –80 oC until analysis. For the measurement of enzyme activity, the larvae were individually homogenized with 1 ml artificial seawater (34 ppt) using a sonicator (Sonifier 150®, Branson Ultrasonics Corporation, Danbury, USA) in an ice-cold bath. The aliquot samples of the same larva were treated with 20 l of enzyme substrate (Boc-Phe-Ser- 9 Arg-MCA) using a repeating pipet and syringe tip (Nichimate Stepper), and then were vortexed well using a tube mixer (Ms1 Minishacker, Kika Works (Asia) Sdn. Bhd., USA) before being placed in an incubator at 37 oC in darkness for 15 minutes. Further, 20 l of 0.5M SDS (sodium dodecyl sulfate) was added to the samples, and they were then centrifuged at 9,000 rpm for 5 minutes at 4 oC (Kubota 6900, Tokyo, Japan). One hundred l of the supernatant of the centrifuged samples and standard fluorescent product were placed into 96 multi-well microplates to measure the tryptic activity, which was determined by the change in the fluorescent product at excitation (360 nm) and emission (460 nm) using a fluorescence multi-well plate reader (Cytofluor Series 4000® , Applied Biosystems, CA, USA). Sterilized artificial seawater (34 ppt) was used as a blank sample for the measurement. 2.3.2 Fatty acid analysis For the analysis of fatty acid composition, P. similis and B. rotundiformis samples were collected during the exponential growth phase and concentrated using a nylon plankton net with a 10-m mesh size. The concentrated rotifer was washed in running tap water to remove salt, rinsed with distilled water, dried from beneath the net using filter paper and stored at –80 oC prior to analysis. The total lipids and fatty acid composition of the samples were analyzed at a commercial laboratory (Chlorella Industry Co., Fukuoka, Japan) following the method of Folch et al. (1957). The rotifer 10 methanolysates were prepared under 100 oC for an hour after the addition of 2M hydrogen chloride methanol. Fatty acid methyl esters (FAME) were extracted by petroleum ether. Gas chromatography analysis was performed using a GC-14A (Shimadzu Scientific Instruments, Inc.) equipped with a HR-SS-10 column. The column temperature was regulated at 150 to 220 oC. Individual fatty acids were quantified by means of the response factor to pentadecanoic acid methyl ester (GL Sciences Inc.) as the internal standard, which was added after the FAME extraction. As a reference, a similar procedure of mass culture and fatty acid analysis was performed with the B. rotundiformis Indonesian strain (Hagiwara et al., 1995; Wullur et al., 2009). 2.4. Statistical analysis The differences between means in food quantity, tryptic activity, growth and survival of the larvae were first analyzed using one-way ANOVA (p<0.05) and further analyzed using the Tukey-Kramer test (p<0.05) if a difference was detected. All data in percentage or ratio terms were arcsine-square-root transformed prior to the analysis. The feeding selectivity of the larvae on two rotifer species in mixed feeding treatments was analyzed using Chesson’s selectivity index (i): i = (ri/pi) /(ri/pi) 11 where ri is the frequency of prey i in the larval gut, and pi is the frequency of prey i in the environment. This index, i, varies between 0 and 1 with i = 0.5 indicating nonselective feeding towards prey i, i > 0.5 indicating a preference for prey i, and i < 0.5 indicating discrimination against prey i. Significant differences in selectivity between two rotifer species were analyzed using Student’s t-test to compare the selection of a specific rotifer species to the natural selection by using the equation in Chesson (1983): t=  i  0.5 s2 / K where i is the sample mean and s2 is the sample variance of the K estimates of i. 12 3. Results 3.1. Ingestion and food selectivity The body size and dry weight of the P. similis and B. rotundiformis used in the present study are presented in Table 2. Mouth opening occurred in the seven-band grouper larvae during the night at 3 DAH, and the mouth size of the larvae at 4 DAH was 18020 µm (n=10). The larvae at 4 DAH showed active feeding on both rotifer species just after the addition of rotifers into the rearing tanks, and all larvae at 4 DAH ingested rotifers in all dietary treatments (100% feeding incidence). The numbers of rotifers in the guts of the larvae were significantly different among treatments except at 10 DAH (ANOVA, p<0.05). The feeding quantity of the larvae in the combined treatment was not significantly different from that of the larvae fed P. similis alone, but it was significantly higher than that of the larvae fed B. rotundiformis alone from 4 to 8 DAH (Tukey-Kramer test, p<0.05, Fig. 1). The larvae co-fed 2 rotifer species significantly selected P. similis over B. rotundiformis at 4 DAH, but the selection shifted to B. rotundiformis from 6 DAH (Student t-test, p<0.05, Fig. 2). 3.2. Digestion The tryptic enzyme activity of seven-band grouper larvae in all dietary treatments was not significantly different except at 10 DAH (ANOVA, p<0.05). On this day (10 DAH), the tryptic enzyme activity of the larvae receiving the co-feeding 13 treatment was significantly higher than in the single-species feeding treatments (TukeyKramer, p<0.05). The tryptic enzyme activities among the larvae in the latter two treatments were not significantly different (Tukey-Kramer, p<0.05) (Fig.3). 3.3 Fatty acid composition Total lipids per wet weight of P. similis fed with the N. oculata NIES-2146 strain and “super fresh” C. vulgaris V-12® were 2.4 and 2.6%, respectively. The fatty acid composition in the total lipids of P. similis changed according to the microalgae. The relative levels of essential fatty acids (EFA) for marine fish larvae such as eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6) in the total lipids of P. similis cultured by the N. oculata NIES-2146 strain were 23.2, 0.0 and 5.3%, respectively, while these were 11.0, 17.5 and 0.5%, respectively, when “super fresh” C. vulgaris V-12® was fed to the rotifers (Table 1). The levels in B. rotundiformis fed “super fresh” C. vulgaris V-12® were 5.8, 6.1 and 1.2%, respectively (Table 1). The ratios of DHA/EPA in two rotifer species fed “super fresh” C. vulgaris V-12® were 1.59 and 1.05 for P. similis and B. rotundiformis, respectively. 14 3.4. Larval rearing The growth of the larvae was significantly different among treatments during the experiment (Fig. 4; ANOVA, p<0.05). The growth of the larvae at 4 to 6 DAH was significantly greater in the combined treatment than in the single-species feeding treatments (Tukey-Kramer, p<0.05). From 8 to 10 DAH, growth was higher in the treatment with B. rotundiformis alone and in the combined treatment than in the treatment with P. similis alone (Tukey-Kramer, p<0.05). The larvae co-fed P. similis and B. rotundiformis showed higher survival (14.3 %) than those fed P. similis alone (2.7 %) at the end of experiment at 10 DAH (Fig. 5). Larvae fed B. rotundiformis alone showed intermediate survival (6.4 %, Fig. 5). 4. Discussion The body size of P. similis in the present study was consistent with the previous report (Wullur et al., 2009). The dry weight of P. similis (35.97.8 ng individual-1) was 6-fold lower than that of B. rotundiformis. The effects of two rotifers on the seven-band grouper larvae in terms of survival, growth and feeding activity were tested based on rotifer density instead of energy content because rotifer density is more informative from a practical perspective for hatchery operators, who usually feed larvae based on rotifer density. 15 Seven-band grouper larvae actively ingested P. similis just after mouth opening at 4 DAH. Thereafter, 100% feeding incidence was observed in all treatments until the end of the experiment. The number of rotifers in the larval gut was higher in the larvae receiving the treatment containing P. similis than in those fed B. rotundiformis alone (Fig. 1). This may be because P. similis is much smaller than B. rotundiformis and the larvae could consume more P. similis to fill the available space in their guts. When 2 rotifer species were co-fed to the grouper larvae, Chesson’s selectivity index indicated that the grouper larvae showed higher selectivity for P. similis at 4 DAH (Fig. 2). This preference became neutral at 5 DAH, and the larvae switched their preference to larger rotifers (B. rotundiformis) after 6 DAH. It has been suggested that size, motion and color (Utne-Palm, 1999; Shaw et al., 2003; Tanaka et al., 2006; Akazawa et al., 2008) are among the characteristics according to which fish larvae select live food. Our observations indicated an apparent similarity in the swimming behavior and color of the two rotifer species (unpublished); thus, it is likely that the cause of the higher prey selection of P. similis by grouper larvae during early feeding (Fig. 2) is attributable to this rotifer’s smaller body size. The change of preference by the larvae to a larger rotifer (B. rotundiformis) after 6 DAH could be to improve feeding efficiency. After fish larvae ingest a food item, digestion must occur to obtain energy for survival and growth. The present study indicated that grouper larvae can ingest, digest and utilize P. similis as an energy source, since larvae fed P. similis grew consistently 16 and survived until 10 DAH (Figs. 4, 5). The tryptic enzyme activity of the larvae, which is one of the appropriate indicators of fish larval digestion (Ueberschar, 1995; Lemieux et al., 1999; Cara et al., 2007), revealed that seven-band grouper could digest P. similis similarly to B. rotundiformis (Fig. 3). Although the grouper larvae ingested greater numbers of P. similis compared with B. rotundiformis during the initial feeding stage (Fig. 1), larval growth was similar between the two treatments (Fig. 4). This was due to the differences in size and biomass of the two prey species. We assume that larvae fed the larger B. rotundiformis obtained more energy per catch than those fed the smaller P. similis. After 8 DAH, the selection of B. rotundiformis by larvae became more active (Fig. 2), resulting in better larval growth (Fig. 4). The fish larvae showed the best growth and survival (to 10 DAH) when they were co-fed the two rotifer species at the same time. In the co-feeding treatment, larvae could encounter prey items with large size variation (80-150 µm in length) and could utilize food items of appropriate size according to their growth level. The inferior growth of the larvae fed P. similis alone after 8 DAH was related to the lower energy content of P. similis in comparison to B. rotundiformis. These results suggest that P. similis can be sufficient for the growth of fish larvae, but should be fed at higher densities than B. rotundiformis. EFAs for marine fish larvae, such as DHA, EPA and ARA (Watanabe et al., 1983; Izquierdo, 1996; Izquerdo et al., 2000; Lie et al., 1997; Rainuzzo et al., 1997; 17 Sargent et al., 1999; Takeuchi, 2001) were manipulated in P. similis by using an enrichment source (Table 1), as has been reported for B. plicatilis by many authors (Whyte et al., 1990; Tamaru et al., 1993; Kobayashi et al., 2005, 2008). The relative proportions of ARA and EPA in the total lipids of P. similis were higher when the N. oculata NIES-2146 strain was fed, while that of DHA and the ratio of DHA/EPA were higher when “super fresh” C. vulgaris V-12® was used. These levels of EFAs and of the DHA/EPA ratio were in the range of the suggested levels for marine fish larvae (Tucker, 1998; Sargent, 1999) From the present study, it was clarified that the euryhaline rotifer P. similis can be used as a live food for rearing fish larvae, including species with small mouth gape such as groupers. 18 Acknowledgements The rotifer Proales similis was collected during the 189th cruise of Kakuyomaru, Faculty of Fisheries, Nagasaki University in July, 2004. We are grateful to the four anonymous referees for their constructive comments for improving this manuscript. The authors express thanks to Russ Shiel for the identification of rotifer species. The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan is gratefully acknowledged for the scholarship awarded to S.W. 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The minute monogonont rotifer Proales similis de Beauchamp: culture and feeding to small mouth marine fish larvae. Aquaculture 293, 62-67. Yamaoka, K., Nanbu, T., Miyagawa, M., Isshiki, T., Kusaka, A., 2000. Water surface tension-related deaths in prelarval red-spotted grouper. Aquaculture 189, 165–176. 26 Figure legends Fig. 1. Food quantity (mean±SD, n=3) of the seven-band grouper larvae fed P. similis at 20 ind./ml (open column), B. rotundiformis at 20 ind./ml (closed column) and mixed rotifers at 20 ind./ml consisting of 10 ind./ml each of P. similis and B. rotundiformis (shaded column). Different letters on the columns indicate significant differences among treatments on the same day (a>b, Tukey-Kramer test, p<0.05). 27 Fig. 2. Chesson’s selectivity index (mean±SD, n=3) of the seven-band grouper larvae for the two rotifer species, P. similis (open squares) and B. rotundiformis (closed squares). Asterisks indicate significant differences between rotifer species at the same age (t-test, p<0.05). 28 Fig. 3. Tryptic enzyme activity (mean±SD, n=3) of the seven-band grouper larvae fed P. similis at 20 ind./ml (open column), B. rotundiformis at 20 ind./ml (closed column) and mixed rotifers at 20 ind./ml consisting of 10 ind./ml each of P. similis and B. rotundiformis (shaded column). Different letters on the columns indicate significant differences among treatments at the same age (a>b, Tukey-Kramer test, p<0.05). 29 Fig. 4. Growth expressed as standard length (mean±SD, n=3) of the seven-band grouper larvae fed P. similis at 20 ind./ml (open square), B. rotundiformis at 20 ind./ml (closed square) and mixed rotifers at 20 ind./ml consisting of 10 ind./ml each of P. similis and B. rotundiformis (gray squares). Different letters on the columns indicate significant differences among treatments at the same age (a>b, Tukey-Kramer test, p<0.05). 30 Fig. 5. Percent survival (mean±SD, n=3) at 10 DAH of the seven-band grouper larvae fed P. similis at 20 ind./mL (20-PS), B. rotundiformis at 20 ind./mL (20-BR) and mixed rotifers at 20 ind./ml consisting of 10 ind./ml each of P. similis and B. rotundiformis (20-PS+BR). Different letters on the columns indicate significant differences among treatments at the same age (a>b, Tukey-Kramer test, p<0.05). 31 Table 1. Total lipids per wet weight (%) and fatty acid composition (%) of P. similis fed Nannochloropsis oculata and “super fresh” Chlorella vulgaris V-12® as well as in B. rotundiformis fed “super fresh” C. vulgaris V-12®. Both rotifers were fed C. vulgaris V-12® prior to being fed to seven-band grouper larvae. Proales similis (%) Brachionus rotundiformis ( %) Nannochloropsis “Super fresh” Chlorella ”Super fresh” Chlorella vulgaris (V-12)® vulgaris (V-12)® 2.4 2.6 1.0 C14:0 7.6 2.6 1.7 C14:1 0.4 0.0 0.0 C16:0 18.8 11.7 18.3 C16:1 12.8 0.9 0.9 C16:2 1.1 6.0 4.7 C18:0 3.6 3.4 4.6 C18:1 9.9 6.0 2.4 C18:2 n-6 3.3 19.7 23.8 C18:3n-3 0.0 4.5 6.1 C20:0 0.3 0.3 0.3 C20:1 1.0 1.0 1.3 C20:4 n-6 5.3 0.5 1.2 C20:5 n-3 23.2 11.0 5.8 C22:0 0.4 0.5 0.3 C22:1 0.2 0.2 1.1 C22:5 n-3 2.4 3.6 3.6 C22:6 n-3 0.0 17.5 6.1 C24:0 0.8 1.3 0.4 C24:1 0.5 0.6 0.4 Others 0.5 8.7 17.0 C22:6 n-3 / C20:5 n-3 0.0 1.59 1.05 Total 100.0 100.0 100.0 oculata Total lipids Fatty acids 32 Table 2. Body size and dry weight of P. similis and B. rotundiformis (meanSD). Species n P. similis 50 B. rotundiformis 50 Body size Dry weight Length Width (ng/ind.;) 80.79.0 35.44.0 35.97.8 148.113.6 115.912.8 247.513.9