The role of different algae in the growth and survival of turbot larvae (Scophthalmus maximus L.) in intensive rearing systems

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1 ICES mar. Sei. Symp., 201: The role of different algae in the growth and survival of turbot larvae (Scophthalmus maximus L.) in intensive rearing systems J. G. Støttrup, K. Gravningen, and N. H. Norsker Støttrup, J. G., Gravningen, K., and Norsker, N. H The role of different algae in the growth and survival of turbot larvae (Scophthalmus maximus L.) in intensive rearing systems. - ICES mar. Sei. Symp., 201: Five species of planktonic algae were tested to examine their effect on growth and survival in turbot larvae reared in static water; the green-water technique". Specific daily growth rates from day 4 to day 18 after hatching ranged from 24% to 39%. Survival to day 18 ranged from 1% to 42%. Growth and survival in turbot larvae were related to the algal species used. The present paper discusses possible effects of this rearing technique, including nutritional effects in terms of fatty acids and amino acids. The role of bacteria in the rearing tanks was examined and it is proposed that the bacteria-controlling function of the algae was more important than their nutritonal effect. The adaptability of the green-water technique in commercial rearing systems is discussed, along with the use of harpacticoids to help maintain tank-wall hygiene. ]. G. Støttrup: Danish Institute fo r Fisheries and Marine Research, North Sea Centre, PO Box 101, DK-9850 Hirtshals, Denmark [tel: (+45) , fax: (+45) ], K. Gravningen: Tinfos Aqua AIS, N-4484 Øyestranda. Present address: Apothekernes Laboratorium A/S, PO Box 158 Skøyen, N-0212 O slo2, Norway. N. H. Norsker: BioProcess ApS, North Sea Centre, PO Box 104, DK-9850 Hirtshals, Denmark. Introduction Despite numerous technical advances in the rearing of juvenile marine fish, the so-called green-water technique is still preferred by many culturists. The maintenance of unicellular algal blooms in larval tanks is considered to be beneficial for many species of fish and are added either as monocultures or polycultures. Few studies have been conducted comparing the effect of different algal species. Howell (1979) stated that the choice of algal species was important and that the best results were obtained when Isochrysis galbana was among the selected species. Likewise, Nash et al. (1974) concluded that the addition of /. galbana gave the best results. The general consensus was that the addition of microalgae served as a direct or indirect nutritional booster for fish larvae (Howell, 1979; Reitan et al., 1991). Scott and Baynes (1979) and Scott and Middleton (1979) further suggested that this effect might be directly related to the lipid content of the algae. Also, several workers have emphasized the requirement for dietary sources of highly unsaturated fatty acids such as eicosapentanoic acid and docosahexanoic acid in marine fish larvae (Cowey et al., 1976; Watanabe, 1982), both of which are generally present in high amounts in most marine planktonic algae. In experiments on Atlantic halibut larvae (Hippoglossus hippoglossus L.), Næss et al. (1990) addressed the same issue and pointed out effects on the light regime as a possible function of the addition of algae. They concluded that this effect was more important than any nutritional effect from the algae. Other effects of the algae can also be hypothesized, such as the release of free amino acids acting as attractants (appetite stimulator). Lately, more attention has been paid to the microbiology in the rearing tanks. It is the opinion of these authors that this aspect of rearing technology should be considered just as important as various nutritional or environmental aspects. The bacteria associated with the intensive culture system, generated primarily from the addition of live food (enriched rotifers and Artemia nauplii), was reported to be one of the destabilizing factors in the production of turbot (Perez Benavente and Gatesoupe, 1988; Person-Le Ruyet, 1989). In the light of this development it is pertinent to focus attention on the algal-bacterial interactions and, more specifically, antibacterial activity of phytoplankton. The presence of antibacterial properties in algae was established several

2 174 J. G. Støttrup, K. Gravningen, and N. H. Norsker ICES mar. Sei. Symp., 201 (1995) decades ago (Pratt, 1942) and marine algal species such as Tetraselmis sp. have been shown to produce antibacterial compounds (Kellam and Walker, 1989). The objective of this study was to examine the role of live phytoplankton and their interaction with the bacterial populations in fish larval rearing tanks. Also intended was examination of the species specificity of the algal effect. The fatty acid and amino acid content of rotifers grazing on the different algae was analysed to examine whether or not the microalgal effect could be attributed to the nutritional value of the residual rotifers in the larval tanks, whose biochemical composition gradually shifts from the original enrichment used to that of algal enriched rotifers. Materials and methods The experiment was carried out at Tinfos Aqua A/S, a commercial turbot rearing hatchery in southern Norway. Eggs and sperm were collected from broodstock kept under a controlled light regime and at a stable temperature (12 C). The fertilized eggs were incubated at 12 C in a flow-through system supplied with air-bubbling to ensure an even distribution of eggs. The temperature was gradually raised to 18 C and 2 days after hatching the larvae were transferred to the experimental rearing tanks. Experimental conditions The experimental rearing tanks were 350 L light-grey GRP with rounded bottoms and central aeration. Continuous illumination was provided by 2 X 4 0 W neon (cool-white) tubes placed cm above the water surface. Ten tanks were filled with temperate (18 C), filtered sea water (lfxm) and one of the following algae at various start concentrations (duplicate treatments): Rhodomonas baltica and Dunaliella tertiolecta ( cells xl_1), I. galbana, clone T-Iso and Pavlova lutheri (300 (il-1) and Chlorella sp. ( (x r1). The algal species were obtained from the Marine Laboratory in Helsingør, Denmark, except for Chlorella sp., which was obtained from a European hatchery. Hereafter, the algae will be referred to by their generic names only. Each tank was stocked with 2500 turbot larvae. The larvae were fed rotifers (Brachionus plicatilis) three times daily at 0800, 1400, and 2000 h, in a feeding regime aimed at minimizing the amount of residual rotifers at the next feeding event. The rotifers were taken from the mass production system, where they were fed Isochrysis and a yeast-based enrichment (including highly unsaturated fatty acids (HUFAs) and vitamins), collected and rinsed in fresh sea water and stored at 12 C for up to 24 h. On day 6, copepodites (NVI-CII) of the harpacticoid Tisbe holothuriae were added to each tank to help maintain the tank wall and bottom free of biofilm. At 8 10 days post-hatching, freshly hatched nauplii of the brine shrimp Artemia salina (AT-1; Brazil strain from ATP, Spain) were fed to the larvae. They were then substituted after a further 2 days by 1-d-old, enriched San Francisco Bay Artemia nauplii. The rotifers and brine shrimp used in this experiment were taken from mass production cultures subjected to routine hatchery treatment (hatch, collection, enrichment, wash). Day references all refer to larval age in days post-hatching. Flow was introduced on day 13 in one Isochrysis and both Dunaliella tanks and on day 15 or 16 in the remaining tanks. Experimental sampling The larvae were sampled on days 4, 8,12,16, and 18 for dry weight analyses. A minimum of 16 larvae (except Dunaliella treatments; 8-16) were sampled from each tank, rinsed in distilled water, transferred to a glass slide and dried at 60 C for 24 h. They were then cooled and stored in a desiccator for subsequent weighing on a Cahn electrobalance with a precision of 0.1 jxg. Samples ( ml) for bacterial counts were taken daily from the central aeration area to get a representative sample from each tank. Samples for fatty acid analysis were taken of 2- and 18-d-old larvae. Samples for rotifer fatty acid and free amino acids were taken from the mass production cultures after 24 h cold storage at 12 C. Potential algal dietary effects on rotifers were investigated in a separate rotifer enrichment experiment using different algal enrichment diets. These rotifers are considered representative of residual rotifers in the larval rearing tanks. Samples of algae-enriched rotifers were sampled after feeding on the specified microalgae for 3h (fatty acid) or 3-12 h (free amino acid) at 18 C. Fatty acid samples were also taken of the algal species. Growth Specific growth rate (SGR%) was calculated from: SGR (%) = 100[(expG) - 1 ], where the instantaneous growth rate (G) was calculated from: G = (InDW, - In DW0)/(T, - Tu), where DW 0 and DW, are the initial (T0) and final (Tt) dry weight. Bacterial counts and identification Bacterial samples were diluted in sterile sea water and spread on Tryptic Soy Broth (Difco) with NaCl (Merck) added to 2% (TSA-2), except on day 6 when Marine Agar (Difco) was used. After 24h of incubation (20 ± 2 C) colony-forming units were registered. Bac

3 ICES mar. Sei. Symp ( 1995) Algae in the growth and survival of turbot larvae 175 teria were identified based on colony and cell morphology, gram staining, motility, growth at 4 and 20 C, growth at 0,3, and 10% NaCl, fermentative metabolism (Hugh Leifson 2% NaCl), production of: catalase, oxidase, gas from glucose, arginine dihydrolase, lysine decarboxylase, and ornithine decarboxylase, sensitivity to 0/129. Data analysis The growth and survival data were subjected to analysis using SYSTAT statistics (Wilkinson, 1990). Multiple variance analysis was performed to test the effect of treatment larval size and survival to day 18. Amino acid samples Triplicate samples were extracted in 6% (final concentration) trichloroacetic acid in cryotubes for at least 24 h before analysis. The analyses were carried out at the University of Bergen, as described in Fyhn (1989). Fatty acid analysis Samples for fatty acid analysis were frozen in liquid nitrogen and stored at -80 C. Lipids were extracted according the method of Bligh and Dyer (1959). Each sample was run twice on a Perkin-Elmer 8310 gas-chromatograph equipped with a flame-ionization detector and a glass column packed with 10% Silar 10C on a Gaschrom Q ( mesh). Nitrogen was the carrier gas at lo.omlmin-1 and column temperature programmed from 195 C to 240 C by 2 Cmin_1. Peak identification was done by comparison with standards containing mixtures of fatty acids in known quantities. Heptadecanoic acid (C17:0) was added to each sample as an internal standard, and used to calculate the amounts of the individual fatty acids. Results Larval growth and survival Larval growth (dry weight) in the 10 tanks is shown in Figs. la -e. Larval survival to day 18, dry weight at age 18 and SGR are given in Table 1 All treatments except Isochrysis showed consistent results in terms of growth, with little variation between duplicates. The largest larvae on day 18 were those in the Isochrysis-1 tank, averaging 3.5 mg and an average daily growth rate from day 4 to day 18 of 39%. In the Isochrysis-2 tank, the larvae grew well until day 12 and were at this time larger than those in the Chlorella and Dunaliella tanks (compare Figs. la, b, and d). However, between day 12 and day 16 their growth rate fell to 11%, and although it again rose to 46% after day 16 the larvae were smaller than those in the other Isochrysis tank by day 18. The larvae in the Pavlova and Rhodomonas tanks (Figs. lc and e) grew well, albeit at lower rates than those in Isochrysis-1, average SGRs of 32-33% and average dry weights around 2 mg at age 18 days (Table 1). On day 18, these larvae and those in both Isochrysis tanks were significantly larger than the larvae in the Dunaliella and Chlorella tanks (see Table 2). The Dunaliella larvae were also significantly larger than the Chlorella larvae. In the Dunaliella tanks the larvae started out as slow growers with the lowest growth rates to day 8; 18-20%. From day 8 to day 16 their growth rates increased to 29% and then to 59-63% during the next 2d (Fig. lb). Despite poorest growth rates in the two Chlorella tanks (average SGR from day 4 to day 18; 24-25%), the best survival was obtained in one of these tanks (42%). A relatively high survival rate was also found in the remaining Chlorella tank (28%). The survival rates for the Rhodomonas tanks were persistently high; 37 and 38% and were significantly higher than the survival rates in the Dunaliella, Pavlova, and Isochrysis treatments. Survival rates in the haptophyte tanks ranged from 19 to 24% and were similar. High mortalities were registered in both Dunaliella tanks, and only 1 and 8% survived. Algal growth in larval tanks Algal growth in the larval tanks is shown in Figs. 2a and b. Dunaliella remained stable, and Isochrysis grew very little, during the experimental period. On the other hand, Pavlova, Rhodom onas, and Chlorella grew slowly throughout the experimental period. Both Chlorella and Rhodomonas peaked at a late stage and a decline in cell numbers was evident from day 12 in Chlorella and days in Rhodomonas. In one of the Pavlova tanks, a peak was observed on day 15, whereas in the other tank a decline in cell numbers was observed on day 12. The ph in the larval tanks increased from 7.8 to by day 8, remaining at this level until cell numbers started to decline, whereupon ph decreased towards the more normal value for sea water. Microbiology The colony forming units were low and stable in the Isochrysis tanks throughout the sampling period, while they were more variable in the other tanks (Figs. 3a-e). The sharp peak in bacterial numbers which was observed in one Rhodomonas tank and both Chlorella and Pavlova tanks on day 6 coincided with the use of Marine Agar as growth medium instead of TSA-2. No data were available on day 7. Bacteria were grouped to Vibrio spp., V. alginolyticus, Pseudomonas spp., and Acinetobacter spp. In

4 1 7 6 J. G. Støttrup, K. Gravningen, and N. H. Norsker i c e s mar. s d. Symp., 201 ( 1995) 3 a b 2 1 o 01 E S D 0, C 4 d 2 3 I 2 3 o 1 0 o e Figure 1. Growth (DW) in turbot larvae from day 4 to day 18 from hatching. One figure for each treatment, a. Dunaliella-, b. Chlorella; c. Pavlova; d. Isochrysis; e. Rhodomonas. Squares and triangles indicate - 1 and - 2 of the duplicate treatments, respectively. Average of 8-20 larvae ± Vvar.

5 ICES mar. Sei. Symp., 201 (1995) Algae in the growth and survival of turbot larvae 111 general, higher diversity of bacteria was observed in the Dunaliella and Chlorella tanks, compared with the other treatments. V. alginolyticus was not observed in the Pavlova and Rhodomonas tanks and Pseudomonas spp. was not observed in the Rhodomonas tanks. Amino acid content The total amount of amino acids was higher in rotifers fed Dunaliella, Rhodomonas, Pavlova, and Isochrysis (15.74 ±2.66, ±0.91, ±1.87, and ± 1.22 nmoles mg WW_1, Table 3) compared with those fed Chlorella, or those taken from the production system [(TA)(8.87, 7.33 ±0.98nm olesm gw W - )]. This difference in content was largely due to higher contents of aspartate, serine, glutamate, glycine, and valine. In Chlorella-fed rotifers, the relatively high content of alanine was compensated by the relatively low contents of several amino acids, such as threonine, serine, proline, valine, leucine, tyrosine, and phenylalanine, four of which are known to be essential to fish. Dunaliella, Chlorella, and Pavlova had relatively high amounts of alanine and low amounts of valine and leucine. Fatty acid content Fatty acid content in the microalgae is given in Table 4.. Unfortunately, the Isochrysis and Pavlova samples were damaged. Trace amounts of essential HUFAs (highly unsaturated fatty acids) were found in Dunaliella, whereas Rhodomonas contained 11% DW EPA (20:5n- 3) and 10% DHA (22:6n-3). In an earlier work (Støttrup and Jensen, 1990) Isochrysis was found to contain lower amounts of EPA (0.6% DW) and higher amounts of DHA (19.5% DW). Chlorella contained very high amounts of 20:5n-3 (38%) and very small amounts of DHA. HUFA content in the algal-enriched rotifers generally reflected that of their diet (Table 5). The production rotifers contained 3 and 4% ind~' of EPA and DHA respectively. The combined EPA and DHA content ind-1 remained unchanged when fed Isochrysis, but the EPA fraction fell to 2%. EPA and DHA content doubled when rotifers were fed Pavlova or Rhodom o nas. No traces of these fatty acids were found in rotifers fed Dunaliella. Rotifers fed Chlorella contained the highest amount of EPA and DHA (8.8 and 1.6%). EPA/ DHA ratio was similar in production Isochrysis and Rhodomonas rotifers and highest (2.2) in Chlorella fed rotifers. Fatty acid distribution in the fish larvae at age 1 and 18 days after hatching is given in Table 6a and b. HUFA levels were high in the 1-d-old larvae (EPA: 5%, DHA: 12-15%). At 18 days of age, differences in the fatty acid distribution were minor within each treatment. Larvae in the Pavlova tanks had the highest content of EPA and DHA, those in the Chlorella tanks the lowest. The EPA/ DHA ratio was relatively similar Discussion Growth and survival Significant effects of different algal species on larval growth and survival were evident in this study. The chlorophyte Dunaliella stands out as a poor treatment for turbot rearing, resulting in poor growth and survival (see Fig. 4), and confirms results obtained elsewhere (Howell, 1979; Scott and Middleton, 1979). The haptophytes Isochrysis and Pavlova were relatively good treatments in terms of survival but growth rates in the Isochrysis tanks varied significantly.. Both good survival and good growth rates were obtained adding R hodom o nas to the tanks, whereas the use of Chlorella resulted in high numbers of small larvae with comparatively low SD (Fig. 4). Within-treatment variation in growth rates was insignificant, except for Isochrysis. Table 1. Survival to day 18, average dry weight (DW) and ± V var for 18-d-old turbot larvae reared in static intensive systems with different algal species. Specific growth rates (SGR) for days and days 4-18 (total SGR) are also given. Treatment Age (days) n Avg. DW (mg) ± Vvar (mg) S G R (%) d Total SGR (%) Survival (%) Dunaliella Dunaliella Chlore lia Chlorella Pavlova-l Pavlova Isochrysis-l Isochrysis Rhodom onas Rhodom onas

6 178 J. G. Støttrup, K. Gravningen, and N. H. Norsker ICES mar. Sei. Symp., 201 (1995) Table 2. Results of the multiple variance analysis for significant differences on the 5% level are given here. Blank cells = not significant. Top-right of dashed cells are p values obtained on the DW data, bottom-left on the survival data. Dunaliella Chlorella Pavlova Isochrysis Rhodomonas Dunaliella _ Chlorella Pavlova _ Isochrysis _ Rhodom onas C t I «««4000 E r». / \ / A * 1 / ' / r v \ \ Age (days from hatching) Age (days from hatching) Figure 2. Algal growth expressed as cell numbers ^ r 1 in the larval tanks during rearing trials with turbot. Final cell count indicates day when water was exchanged and flow introduced, a. Isochrysis-1 (circle), Isochrysis-2 (square), Pavlova-1 (triangle up), Pavlova-2 (triangle down), Rhodomonas-1 (diamond), Rhodomonas-2 (six-sided), b. Dunaliella-1 (diamond), Dunaliella-2 (triangle down), Chlorella-1 (triangle up), Chlorella-2 (square). Improved growth and survival have previously been attributed to the addition of unicellular algae, especially Isochrysis, to larval tanks either as a monoculture or together with other species (Jones, 1970; Howell, 1979). Reitan etal. (1991) showed ingestion and assimilation of Tetraselmis sp. by yolk-sac larvae of halibut (Hippoglossus hippoglossus L.) and van der Meeren (1991) found evidence of filter feeding on algal cells in cod (Gadus morhua L.) larvae, although the amount ingested was estimated to be insufficient to support growth, and other external sources of food were required. Turbot larvae drink water to maintain their water balance (Korsgård, 1991) and algae may thus be ingested passively and perhaps of direct nutritional importance during firstfeeding. Thus, all the larvae except those in the Dunaliella tanks would be expected to show higher growth and survival. This was not the case, as shown in Table 2. Scott and Baynes (1979) found that dead algae, whether frozen or dried, were as effective as live algae in improving the survival of turbot larvae, implying indirect nutritional effect of the algae added to the tanks. This was confirmed by Scott and Middleton (1979), who obtained poorer growth during the rotifer feeding stage for turbot larvae reared with Dunaliella added to the tanks compared to larvae in tanks with Isochrysis, Phaeodactylum, or Pavlova, and suggested this to be due to the lack of HUFAs in Dunaliella. In the present experiment, the rise in ph in all the tanks indicated algal growth, although algal cell counts did not increase in all the tanks, possibly due to intense grazing by the rotifers. Also, high feeding activity in the rotifers was observed immediately after transfer to the fish larval tanks. Thus, the indirect nutritional effect of the algal addition through residual rotifers, the fraction of the live-feed additions not immediately consumed, was examined indirectly through a separate experiment whereby the rotifers were allowed to feed on monoalgal diets. The rotifer HUFA content reflected that of their diet after 3 h of feeding at 18 C (Table 5). A significant fraction of the larval rotifer intake will consist of rotifers that have been in the larval tank for more than 3h. Thus, differences in the rotifer profile after having fed on the phy-

7 ICES mar. Sei. Symp., 201 (1995) a A Igae in the g ro w th a n d su rv iv a l o f tu rb o t larvae 179 b ihv ~r o 70 0 = s «ro «30 1 «> A l i V S e from hatching (days) 100 T 90 -I 80 o o 70 ' IT 60 g s 50 o ro Ï 40 j reco Figure 3. Bacteria numbers (CFU ml-1) in the larval tanks during rearing trials using different algal species. One figure for each treatment, a. Dunaliella; b. Chlorella; c. Pavlova; d. Isochrysis; e. Rhodomonas. Squares and triangles indicate - 1 and - 2 of the duplicate treatments, respectively.

8 180 J. G. Støttrup, K. Gravningen, and N. H. Norsker ICES mar. Sei. Symp (1995) toplankton would be expected to influence turbot larval growth and survival. Sorgeloos et al. (1988) reported strong correlations between dietary content of EPA and growth and DHA and survival in sea bass (Dicentrarchus labrax L.) larvae. Certainly, the total lack of n-3 HUFAs in Dunaliellaenriched rotifers may have been a major factor contributing to poor growth and survival in this experiment, but this relationship was not as clear in the other treatments. EPA content was highest in rotifers fed Chlorella and turbot larvae from one of these two tanks showed the best survival (42%). EPA content in Pavlova and Rhodomonas fed rotifers was similar, yet survival in the Rhodomonas tanks was significantly higher than in the Pavlova tanks (Table 2). DHA in Rhodomonas fed rotifers was highest and larval survival in these tanks was significantly higher than for larvae from the Pavlova, Isochrysis, or Dunaliella tanks. This finding supports earlier results that improved levels of DHA in the live prey organisms significantly improved survival (Støttrup and Attramadal, 1992), although this was found to be significant for the combined rotifer and Artemia feeding stage. In that study a low HUFA content in the rotifer did not affect growth and survival so long as nutritionally adequate Artemia were subsequently provided. The Artemia nauplii fed after days were not Table 3. Amino acid content (nmol/mg WW) in rotifers from the production system (0 H ) and after feeding on different algal species: Dun = Dunaliella, Chi = Chlorella, Pav = Pavlova, Iso = Isochrysis, Rho = Rhodomonas. Average of double samples. Treatment Amino acid 0 H Dun Chi Pav Iso Rho Phs Tau Asp Thr Ser Glu Gin Pro Gly Ala Aba Val Met He Leu Tyr Phe His Lys Arg Total Table 4. Fatty acid content (n.g mg D W 1) in three algal species. Rho = R hodom onas, Chi = Chlorella, Dun = Dunaliella. a-g are unidentified fatty acids. Fatty acids in parentheses not identified by standards but from their position. Fatty acid Dun Chi Rho 14: , : : : a b c d : : e : :3n :3n (18:4) f : g : : :3n :4n :3n (20:4n-3) :5n :0 O.O(K) : :4n :6n : Ratio 20:5n-3/22:6n examined for HUFA content. Initial HUFA levels would have been similar and high due to the enrichment used (Super Selco, see Støttrup and Attramadal, 1992) but may have shifted towards reflecting that of their algal diet, as was the case for rotifers. Thus, dietary effects similar to that of the rotifers could be assumed. Daily growth rates obtained in this study were high and averaged between 24% and 39% from day 4 to day 18. These growth rates were comparable to those obtained in extensive systems (days 4-24 or 31; 22-40%, calculated from Paulsen and Andersen, 1989; days 2-37; 32-36%, Danielsen et al., 1990) and intensive systems (days 2-24; 34%, Olesen and Minck, 1983). Despite trace or no values for EPA and DHA content in rotifers fed Dunaliella, the larvae from the Dunaliella tanks were significantly larger by day 18 than those in the Chlorella tanks. DHA content in rotifers fed different algae, excluding Dunaliella, ranged from 0.42 to 0.62 (xg ind~' (Table 5) and was lowest in those from the Chlorella and Isochrysis tanks and highest in the Pavlova and Rhodomonas tanks. This pattern was also reflected

9 ICES mar. Sei. Symp (1995) Algae in the growth and survival o f turbot larvae 181 in the DHA content in the larvae. However, the larvae from the Pavlova, Isochrysis, and Rhodomonas tanks were significantly larger than those from the Chlorella tanks. Thus, no clear relationship could be demonstrated between dietary EPA and larval growth and survival. With the exception of the Isochrysis tanks, algal D H A may have enhanced larval growth up to an algal-enriched rotifer DHA content of <0.5 xg DHA rotifer1. It should be noted, however, that, in relation to the above-mentioned enhanced growth, these fish larval growth rates did not exhibit significant differences at the 5% level (Table 2). D H A was demonstrated to be far superior as a growth-promoter for juvenile striped jack (Pseudocaranx dentex) (Watanabe et al., 1989). Also, Witt et al. (1984) attributed improved larval growth in turbot to high DH A content in the copepods whose EPA/DHA ratio was between 1.5 and 3.2. In this study the EPA/ D H A ratios varied from 0.65 to 2.22 in the residual rotifers, except those fed Dunaliella, which lacked both EPA and DHA (Table 5). The highest ratio (2.2) was in the rotifers fed Chlorella, and larvae from this treatment showed poorest growth. There was no evidence of any effect of the EPA/DHA ratio on growth or survival in this study. Fyhn (1989) proposed that free amino acids were an important energy source for marine fish larvae, a decrease in the larval free amino acid pool being observed during the yolk-sac stage in Atlantic halibut and cod (Gadus morhua). In turbot larvae, whose yolk contains an oil globule, resorption of free amino acid occurred primarily during the egg stage, providing the major part of energy requirements during this stage (Rønnestad et a l., 1992). These authors also found that during the yolksac stage the energy substrate switched to lipids, while the remaining free amino acids were mainly polymerized into body proteins. By the onset of first-feeding, 90% of the energy requirements was provided by the oil globule. They concluded that lipids derived from the oil globule were the main energy substrate after hatching. Thus, the free amino acid pool is depleted by the end of the yolk-sac stage. Beyond the yolk-sac stage, dietary free amino acid may therefore be critical only to protein synthesis, thus influencing growth. In this study, Chlor- <?//a-enriched rotifiers contained the lowest amount of total free amino acids and a lower content of essential free amino acids such as threonine, proline, valine, and leucine (Table 3). Dunaliella-ennched rotifers also contained low amounts of valine and leucine compared with those enriched with Isochrysis and Rhodomonas (Table Table 5. Fatty acid content (pug ind ') in rotifers from the mass production unit (rotifers), or fed different algal species (R + species). Iso = Isochrysis, Dun = Dunaliella, Rho = Rhodom onas, Chi = Chlorella, Pav = Pavlova, a and b are unidentified fatty acids. Fatty acids in parentheses not identified by standards but from their position. Fatty acids Rotifers R + Dun R + Chi R + Pav R + Iso R + Rho 14: : (X) : : : : : :3n :3n (18:4) a : : b 0.00 O.(K) : :3n :4n :3n (20:4n-3) :5n : : :4n :6n : Ratio 20:5n-3/22:6n

10 182 J. G. Støttrup, K. Gravningen, and N. H. Norsker ICES mar. Sei. Symp (1995) Table 6a. Fatty acid content in 1-d-old (jjig ind~') and 18-d-old turbot larvae ( xg mg DW ') from the different treatments. Dun = Dunaliella, Chi = Chlorella, Pav = Pavlova ( - 1, - 2 = duplicate treatments), a = unidentified fatty acid. Fatty acids in parentheses not identified by standards but from their position. Fatty acid 1-day 1-day Dun-1 Chl-1 Chl-2 Pav-1 Pav-2 14: : : : : : : :3n :3n (18:4) : : a : :3n :4n :3n (20:4n-3) :5n : :4n trace :6n : Ratio 20:5n-3/22:6n Table 6b. Fatty acid content in 18-d-old turbot larvae (pig mg D W 1) from the different treatments. Iso = Isochrysis, Rho = Rhodomonas. (-1, -2 = duplicate treatments), a = unidentified fatty acid. Fatty acids in parentheses not identified by standards but from their position. Fatty acid Iso-1 Iso-2 Rho-1 Rho-2 14: : : : : : : :3n :3n (18:4) : : a : :3n :4n :3n (20:4n-3) :5n : :4n :6n : Ratio 20:5n-3/22:6n ). As the larval growth in the Dunaliella and Chlorella tanks was significantly lower than in the other tanks, it cannot be excluded that low levels of essential free amino acids in the rotifers in these tanks were a limiting factor for growth in turbot larvae. On the other hand, whereas even lower contents of valine and leucine were found in rotifers fed Pavlova than in those fed Dunaliella, larval growth in the Pavlova tanks was similar to that in the Rhodomonas tanks. The results obtained in this experiment do not therefore support the hypothesis of a clear relationship between growth in turbot larvae and free amino acid content in residual rotifers. Microbiology In routine measurements of sea-water quality parameters, carried out by an intensive hatchery, large seasonal differences in the bacterial growth potential in the raw sea water were observed (Gravningen, unpubl. data). Sea-water samples incubated in the dark over several days showed a typical peak in colony-forming units after 3-4 days. The peak varied by 2 log values, being consistently high in the spring and autumn. These periods were also characterized by poor results in the larval rearing and the positive effect of the addition of algae was then very pronounced. A possible interpretation of this phenomenon was that antibacterial sub

11 i c e s mar. Sei. Symp., 201 (1995) Algae in the growth and survival of turbot larvae 183 Table 7. Larval dry weight (DW), survival rate, DHA and EPA content and EPA/DHA ratio on day 18 and DHA and EPA content in algal-enriched rotifers. Algal species in larval tanks Larval DW day 18 (mg) DHA in residual rotifers DHA in larvae day 18 Larval survival to day 18 EPA in residual rotifers EPA in larvae day 18 EPA/DHA residual rotifers EPA/DHA larvae day 18 Dunaliella _ 1.58 Dunaliella Chlorella Chlorella Pavlova Pavlova Isochrysis Isochrysis Rhodom onas Rhodom onas E g Q Survival (%) to day 18 from hatching Figure 4. Turbot larval DW (±V var) and survival to day 18 from hatching. Larvae were reared in intensive systems using static water and five different species of monoalgal cultures. Dunaliella (circle), Chlorella (square), Pavlova (triangle up), Isochrysis (triangle down), and Rhodomonas (diamond). stances produced by the microalgae in the larval tanks were somehow controlling bacterial growth and, thus, improving conditions for larval growth and survival. Antibacterial properties of microalgae were demonstrated several decades ago, Pratt (1942) extracting antibacterial substances from Chlorella and showing them to inhibit growth in both gram-positive and gram-negative bacteria. Several marine planktonic algae, such as Tetraselmis sp., have been shown to produce antibacterial compounds (Kellam and Walker, 1989) and inhibition of bacterial fish pathogens by Tetraselmis suecica was demonstrated by Austin et al. (1992). In another experiment (Gravningen and Støttrup, unpubl. data), a possible antibacterial effect of Rhodomonas and Isochrysis was tested on monocultures of V. anguillarum serotype 01 in an in vivo dialysis experiment. Bacterial numbers as indicated by CFU counts on TCBS medium showed a sharp decline (1-2 log) from day 2 of the experiment, compared to controls. In the present experiment, the bacterial numbers in all the larval tanks were less than CFU ml-1. The CFUs in the duplicates were similar and the differences between treatments were less than 1 log, thus per se insignificant (Fig. 3). However, the diversity and species of bacteria flourishing in the tanks varied between treatments. The Dunaliella and Chlorella tanks which were characterized by low larval growth rates (Fig. 4) showed high CFU and a generally higher bacterial species diversity. In the haptophyte tanks, which were associated with higher larval growth rates (except for one Isochrysis tank), low bacterial species diversity and low CFU prevailed. Larval survival, on the other hand, seemed not to reflect microbiological data. A low bacterial species diversity may be the result of both bacterial interspecific competition and species-specific antibacterial activity of the different algal species. Gatesoupe (1989) found a positive correlation between the proportion of V. alginolyticus in the turbot flora and survival rate and suggested competition with opportunistic bacteria as a possible explanation. In the present study, V. alginolyticus was found in low numbers in the Dunaliella, Chlorella, and Isochrysis tanks and not isolated from the water in larval rearing tanks with the highest average survival (Rhodomonas tanks). In all treatments on days 2 and 5, platings from larval surface resulted in monocultures of Vibrio spp. (not V. alginolyticus). Similar microbiological results were obtained in previous and later studies (Gravningen, unpubl. data), implying a generalized picture of specific colonization of the larval surface during the early stages (until day 5), followed by an

12 184 J. G. Støttrup, K. Gravningen, and N. H. Norsker ICES mar. Sei. Symp., 201 (1995) increasing diversity with age, despite the antibacterial effect of the mucus against the genus Vibrio spp. (Fouz et al., 1990). Flow was introduced when the algal culture stopped growing or a collapse was suspected. Previously, high larval mortalities had been experienced if the water was not changed at this point. This observation stresses the importance of maintaining the algae in the larval tanks alive and growing. The result in the Isochrysis-2 tank probably demonstrates neglect of this point. The larvae grew as rapidly as in the Isochrysis-1 tank until day 12 (Fig. Id), then the algal culture started to collapse on day 12 and the water was exchanged on day 13 (see Fig. 2a). Between day 12 and day 16, SGR in the Isochrysis-1 tank decreased to 10.4%. From day 16 to day 18 the Isochrysis-l larvae had regained larval growth rates similar to those in Isochrysis-2; on average, 38% and 36%, respectively (Table 1). The use of harpacticoids The use of harpacticoids as tank-cleaners for larval rearing is a novel role for copepods in aquaculture, as discussed by Støttrup (1992). The concept, however, is not a novel one, as the same harpacticoid species was used for similar purposes in cultures of other marine organisms by Uhlig (1965). Early copepodite stages were added to the larval tanks, since the copepodite stages are predominantly benthic and remain on the tank walls and bottom, where presumably they are unavailable as food for the turbot larvae. By the time these copepodites attained maturity and began producing nauplii, which are largely pelagic in their early stages, these nauplii would have been too small (newlyhatched; 60 jjun) to interest the fish larvae, which by then were feeding on the much larger Artemia nauplii. It is very unlikely, therefore, that the presence of Tisbe in the tanks might have mediated any nutritional effect of the algae, but they may have helped to prevent bacterial film on the tank walls from accumulating algae, thereby improving the performance of the added algae. Other possible explanations of the observed effects Næss et al. (1990) argued that the effect of the algal addition on the light regime may be an important factor increasing turbidity of the medium and decreasing the visual range of the larvae and subsequent anti-predator behaviour/stress. This, however, would not explain difference in the present results between treatments. Næss et al. (1990) mentioned a higher feeding incidence in halibut larvae reared in green water than in those in clear water. The possible role of leakage of certain free amino acids acting as feeding stimulants has not been considered, but may also be a factor influencing growth in fish larvae. Adaptability to commercial rearing systems An easily applicable method for improving both larval growth and survival has been demonstrated in this study. Since dead or dying algae would merely increase the bacterial substrate, the use of microalgae in larval rearing tanks requires stronger illumination than generally used in clear water systems to ensure that the algae are growing, albeit at slow rates, in the larval tanks. As the algae deplete internal and external nutrient sources, they naturally cease growing and a water exchange is necessary. It is essential that the water is exchanged before the algal culture collapses, otherwise profound effects on larval growth and survival may be expected. The addition of small quantities of nutrients has been tried but generally did not prolong the duration of the static water stage. The addition of Tisbe holothuriae together with the algae has been shown to be of benefit, as these maintain tanks walls and bottom free of biofilm (Støttrup, 1992 and the present study). The suppression of the biofilm formation probably helped to maintain the algal blooms in the larval tanks for prolonged periods. In conclusion, growth and survival in turbot larvae was affected by the algal species in the static system used in this experiment. However, the significant differences observed between algal species was not correlated to any single factor among those examined. The results suggest that the nutritional value of the algae may play a role if that of the species used is particularly poor, as in for example Dunaliella, while interactions between algae and bacteria in the larval tanks may be important in other situations. Recently, more attention has been diverted to microbiology in the larval tanks. Rotifers have been suggested as the source of infection in the production of red sea bream (Pagrus major) (Iwata et al., 1978) and turbot (Nicolas et al., 1989). Antibacterial treatment of rotifers prior to feeding improved rearing results in turbot (Gatesoupe, 1989). More recently, attention has been directed towards the use of probiotics, with the intention of promoting benevolent or harmless bacterial flora in the larval tanks, thereby attempting to outcompete harmful flora. Nicolas and Joubert (1986) reported a 50% reduction in the number of Vibrio spp. and Aeromonas spp. by adding Pseudomonas sp. to the rotifer cultures. Also, the probiotic treatment improved growth of the rotifer culture as well as its dietary value (Gatesoupe et al., 1989). It appears that unless the bacterial flora in the larval tanks is controlled it is likely to be accompanied by high larval mortality and poor performance. Consequently, both prophylactic and therapeutic

13 ICES mar. Sei. Symp., 201 (1995) Algae in the growth and survival of turbot larvae 185 use of antibiotics is widespread in commercial hatcheries for marine fish and shellfish (Nicolas et al., 1989; Brown and Tettelbach, 1988). Brown and Tettelbach, working with vibrio strains, stressed the danger of introducing multiple antibiotics resistance in marine bacteria and the possibility of transfer to human pathogens. In a review on hatchery rearing of turbot larvae, Person-Le Ruyet (1989) suggested interrupting production for some time when pathological problems arose as preferable to the routine use of antibiotics. A better understanding of tank microbiology is highly desirable and, in the light of the present findings, a more detailed study of the antibacterial effects of adding algae to the fish larval tanks. These results demonstrate enhancement effects on both growth and survival in larval turbot by the addition of microalgae, the effect depending on the algal species added. 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