ICES mar. Sei. Symp., 201: 173-186. 1995

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. 1995. The role o f different algae in the growth and survival of turbot larvae (Scophthalmus maximus L.) in intensive rearing systems. - ICES mar. Sei. Symp., 201: 173-186.

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 o f fatty acids and amino acids. The role o f 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, P O Box 101, DK-9850 Hirtshals, Denmark [tel: (+ 4 5 ) 98 94 45 00, fax: (+ 45) 33 96 32 60], K . Gravningen: Tinfos A qua AIS, N-4484 Øyestranda. Present address: Apothek- ernes Laboratorium A/S, P O Box 158 Skøyen, N-0212 O s lo 2, Norway. N. H. Norsker: BioProcess A pS, 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 o f the algae. Also, several workers have emphasized the requirement for dietary

sources of highly unsaturated fatty acids such as eicosa- pentanoic 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 (Hippoglos-

sus 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

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-bub- bling 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 4 0 -5 0 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): R hodom onas baltica and Dunaliella tertiolecta (100-200 cells |xl_1), I. galbana , clone T -Iso and Pavlova lutheri (300 (il-1) and Chlorella sp. (800-1000 ( x r 1). 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 (H U FA s) and

vitamins), collected and rinsed in fresh sea water and

stored at 12°C for up to 24 h. On day 6, 75 000 copepo-

dites (NVI-CII) of the harpacticoid Tisbe holothuriae were added to each tank to help maintain the tank wall and bottom free o f 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 ,1 2 ,1 6 , 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 (50-100 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 DW 0)/(T, - Tu), where D W 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 2 4 h o f incubation (20 ± 2°C) colony-forming units were registered. Bac­

ICES mar. Sei. Symp.. 201 ( 1995) Algae in the growth and survival o f 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.

A m ino 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-chro- matograph equipped with a flame-ionization detector and a glass column packed with 10% Silar 10C on a Gaschrom Q (100-200 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 o f the individual fatty acids.

Results

Larval growth and survival

Larval growth (dry weight) in the 10 tanks is shown in

Figs. l a - 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 Rhodom onas 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, P avlova , 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, R hodom 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 days11-13 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 8 .5 -8 .7 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. 3 a -e ) . The sharp peak in bacterial numbers which was ob­served in one Rhodom onas 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. alginolyti-

cus, Pseudomonas spp., and Acinetobacter spp. In

1 7 6 J. G. Støttrup, K. Gravningen , and N. H. Norsker i c e s mar. s d . Symp., 201 ( 1995)

a3

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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. P avlova ; d. Isochrysis; e. Rhodom onas. Squares and triangles indicate - 1 and - 2 of the duplicate treatments, respectively. Average of 8 -2 0 larvae ± Vvar.

ICES mar. Sei. Symp., 201 (1995) Algae in the growth and survival o f turbot larvae 111

general, higher diversity o f 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.

A m in o acid content

The total amount of amino acids was higher in rotifers

fed Dunaliella, R hodom onas, Pavlova, and Isochrysis

(15.74 ± 2 .6 6 , 12.70 ± 0 .9 1 , 13.77 ± 1 .8 7 , and11.45 ± 1.22 nmoles mg W W _1, Table 3) compared with those fed Chlorella, or those taken from the production system [(TA)(8.87, 7.33 ± 0 .9 8 n m o le s m g W 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 o f 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 o f 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 o f essential H U F A s (highly unsaturated fatty acids) were found in Dunaliella , whereas Rhodomonas contained 11% D W EPA (20:5n- 3) and 10% D H A (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 D H A (19.5% DW ). Chlorella contained very high amounts of 20:5n-3 (38%) and very small amounts of D H A .

H U F A 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 D H A respectively. The combined EPA and D H A content ind-1 remained unchanged when fed Isochrysis, but the EPA fraction fell to 2%. EPA and D H A content

doubled when rotifers were fed Pavlova or R hodom o­nas. No traces of these fatty acids were found in rotifers fed Dunaliella. Rotifers fed Chlorella contained the highest amount of EPA and D H A (8.8 and 1.6%). EPA/ D H A 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. H U F A

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 D H A , those in the Chlorella tanks the lowest. The EPA/ D H A ratio was relatively similar 1-1 .6 .

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 hapto-

phytes 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 h odom o­nas 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 (D W ) and ± V var for 18-d-old turbot larvae reared in static intensive systems with different algal species. Specific growth rates (SGR) for days 16-18 and days 4 -1 8 (total SGR) are also given.

TreatmentAge

(days) nAvg. DW

(mg)± V var

(mg)S G R (%) d 16-18

Total SGR

(%)

Survival(%)

Dunaliella-1 18 16 1.13 0.49 63.2 30.1 7.79

Dunaliella-2 18 9 1.01 0.44 59.3 30.0 1.23

Chlore lia-1 18 24 0.77 0.23 24.6 24.6 42.28

Chlorella-2 18 16 0.81 0.17 14.4 23.5 27.55

Pavlova-l 18 16 1.95 0.66 28.2 33.2 18.78

Pavlova-2 18 16 2.05 0.75 22.7 32.4 24.39Isochrysis-l 18 16 3.49 0.77 43.5 38.7 19.13

Isochrysis-2 18 16 1.11 0.45 46.1 26.8 22.78

R hodom onas-1 18 13 2.01 0.75 27.5 33.3 38.15R hodom onas-2 18 16 1.92 0.95 21.3 31.5 36.82

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 o f dashed cells are p values obtained on the D W data, bottom-left on the survival data.

Dunaliella Chlorella Pavlova Isochrysis Rhodom onas

Dunaliella _ 0.000 0.000 0.000 0.000Chlorella - 0.000 0.000 0.000Pavlova _

Isochrysis 0.048 _R hodom onas 0.010 0.031 0.014 -

3500

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E 3000

2000

1000

r ». / \/ A *

1 / '

/ r v \ \ .

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Age (days from hatching)

14 16 18

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), P avlova-1 (triangle up), Pavlova-2 (triangle down), Rhodomonas-1 (diamond), Rhodom onas-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 (H ippoglos- sus 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 o f 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 first- feeding. Thus, all the larvae except those in the D una­liella 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 H U F A s 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 o f 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 H U F A 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 o f rotifers that have been in the larval tank for more than 3h . Thus, differ­ences in the rotifer profile after having fed on the phy-

ICES mar. Sei. Symp., 201 (1995)

a

A Igae in th e g ro w th a n d s u r v iv a l o f tu rb o t la rv a e 179

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Figure 3. Bacteria numbers (CFU m l-1 ) in the larval tanks during rearing trials using different algal species. One figure for each treatment, a. Dunaliella; b. Chlorella; c. P avlova ; d. Isochrysis; e. Rhodom onas. Squares and triangles indicate - 1 and - 2 of the duplicate treatments, respectively.

180 J. G. Støttrup, K. Gravningen, and N. H. Norsker

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 D H A

and survival in sea bass (Dicentrarchus labrax L.) larvae. Certainly, the total lack of n-3 H U FA s in Dunaliella- enriched 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 Rho­dom onas fed rotifers was similar, yet survival in the R hodom onas tanks was significantly higher than in the

Pavlova tanks (Table 2). D H A in Rhodom onas 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 D H A in the live prey organisms significantly improved survival (Støttrup and Attramadal, 1992), although this was found to be

significant for the combined rotifer and Artem ia feeding stage. In that study a low H U F A 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 10—12 were not

Table 3. Am ino 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 = Rhodom onas. Average of double samples.

TreatmentAminoacid 0 H Dun Chi Pav Iso Rho

Phs 0.86 1.07 1.00 1.01 0.65 0.83Tau 1.56 1.26 0.86 1.38 1.00 1.14Asp 0.26 0.58 0.26 0.59 0.62 0.71Thr 1.12 0.31 0.09 0.26 0.40 0.43Ser 0.57 1.10 0.39 1.29 0.97 1.33Glu 0.68 1.35 0.69 1.34 1.28 1.56Gin 0.00 0.00 0.00 0.00 0.00 0.00Pro 0.52 0.60 0.17 0.64 0.23 0.58Gly 0.36 0.89 0.34 0.91 0.62 0.80Ala 0.39 4.84 2.74 3.29 1.20 1.13Aba 0.04 0.07 0.03 0.06 0.04 0.06Val 0.14 0.40 0.09 0.24 0.76 0.77Met 0.03 0.00 0.00 0.01 0.00 0.00He 0.16 0.22 0.11 0.14 0.28 0.28Leu 0.26 0.39 0.12 0.27 0.69 0.60Tyr 0.50 0.57 0.16 0.47 0.58 0.59Phe 0.19 0.25 0.06 0.12 0.18 0.12His 0.00 0.00 0.00 0.00 0.00 0.00Lys 0.42 0.43 0.53 0.33 0.64 0.56Arg 0.66 1.40 1.24 1.41 1.30 1.21

Total 8.72 15.74 8.87 13.77 11.45 12.70

ICES mar. Sei. Symp.. 201 (1995)

Table 4. Fatty acid content (n.g mg D W “ 1) in three algal species. Rho = R h odom on as, 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

14:0 2.610 3,394 3.82514:1 0.462 0.541 0.22416:0 13.148 7.765 5.58616:1 1.103 13.944 0.961a 0.155 0.000 0.884b 3.090 0.372 0.000c 13.206 0.000 0.000d 0.277 0.105 0.16418:0 0.343 0.177 0.06018:1 3.693 2.298 0.000e 6.040 0.000 2.40218:2 4.379 2.606 0.63118:3n-6 3.538 0.799 0.98818:3n-3 0.000 0.000 7.384(18:4) 31.726 0.232 0.000f 1.146 0.080 14.61820:0 0.137 0.000 0.000

g 0.033 0.000 0.16420:1 1.149 0.177 0.07720:2 0.118 0.000 0.00020:3n-6 0.045 0.182 0.02020:4n-6 0.000 2.917 0.41120:3n-3 0.164 0.000 0.000(20:4n-3) 0.039 0.000 0.38720:5n-3 0.082 22.605 4.98522:0 O.O(K) 0.000 0.00022:1 0.115 0.542 0.20922:4n-6 0.000 0.396 0.00022:6n-3 0.084 0.104 4.43724:0 0.041 0.273 0.000

Ratio 20:5n-3/22:6n-3 1.0 — 1.1

examined for H U F A content. Initial H U FA 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 -2 4 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 D H A content in rotifers

fed Dunaliella, the larvae from the Dunaliella tanks were significantly larger by day 18 than those in the Chlorella tanks. D H A 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 Rhodom onas tanks. This pattern was also reflected

ICES mar. Sei. Symp.. 201 (1995) Algae in the growth and survival o f turbot larvae 181

in the D H A 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 D H A content of < 0 .5 |xg D H A 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 (Pseudocar- anx dentex) (Watanabe et al., 1989). Also, Witt et al. (1984) attributed improved larval growth in turbot to high D H A content in the copepods whose E PA /D H A 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

E PA and D H A (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 E PA /D H A 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 yolk-

sac 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 = R hodom 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

14:0 0.24 0.20 0.41 0.44 0.78 0.3114:1 0.00 0.00 0.(X) 0.00 0.00 0.00

16:0 0.94 0.89 1.31 1.02 1.68 1.0916:1 0.82 0.00 1.69 1.40 1.06 0.9818:0 2.02 1.03 0.96 0.64 4.15 0.9818:1 1.49 0.00 2.06 2.09 1.73 1.9718:2 0.53 0.01 0.82 0.71 0.74 0.9218:3n-6 0.84 0.80 1.15 0.88 0.84 1.01

18:3n-3 0.12 0.00 0.21 0.21 0.34 0.74(18:4) 0.07 0.01 0.11 0.12 0.58 0.36

a 0.00 0.00 0.00 0.00 0.10 0.0020:0 0.06 0.03 0.04 0.04 0.00 0.04

20:1 0.32 0.00 0.42 0.42 0.31 0.41

b 0.00 O.(K) 0.09 0.10 0.13 0.15

20:2 0.07 0.00 0.06 0.06 0.06 0.0920:3n-6 0.04 0.00 0.05 0.05 0.03 0.05

20:4n-6 0.06 0.00 0.16 0.08 0.03 0.07

20:3n-3 0.00 0.00 0.00 0.00 0.01 0.08

(20:4n-3) 0.14 0.01 0.23 0.21 0.14 0.25

20:5n-3 0.26 0.00 1.04 0.58 0.28 0.49

22:0 0.00 0.04 0.00 0.00 0.00 0.0022:1 0.24 0.00 0.31 0.30 0.00 0.31

22:4n-6 0.00 0.00 0.00 0.00 0.00 0.00

22:6n-3 0.33 0.00 0.47 0.52 0.42 0.62

24:0 0.08 0.03 0.19 0.15 0.10 0.16

Ratio 20:5n-3/22:6n-3 0.79 — 2.22 1.13 0.65 0.79

182 J. G. Støttrup, K. Gravningen, and N. H. Norsker ICES mar. Sei. Symp.. 201 (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. D un = 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

14:0 0.29 0.25 1.78 1.04 2.36 1.07 0.9814:1 0.00 0.00 0.00 0.00 0.00 0.00 0.0016:0 0.43 0.61 12.71 6.68 10.33 12.93 11.6816:1 0.25 0.33 3.67 1.72 2.38 3.39 3.2618:0 0.15 0.17 6.64 3.56 5.24 7.37 6.4418:1 0.47 0.74 25.66 14.79 13.91 29.36 26.6818:2 0.08 0.10 5.21 2.64 6.21 4.96 4.9118:3n-6 0.00 0.20 4.51 3.08 0.00 2.51 2.3718:3n-3 0.06 0.06 16.05 7.92 5.02 14.53 15.57(18:4) 0.08 0.07 1.84 0.84 0.99 1.55 1.8020:0 0.03 0.02 0.45 0.18 0.26 0.39 0.3120:1 0.12 0.18 1.63 0.78 1.01 1.81 1.59a 0.06 0.09 0.88 0.00 1.61 0.40 0.3620:2 0.02 0.03 0.85 0.40 0.62 0.88 0.8020:3n-6 0.01 0.01 0.36 0.11 0.13 0.38 0.3120:4n-6 0.03 0.06 1.53 0.98 1.47 1.70 1.7020:3n-3 0.00 0.00 2.50 1.10 1.09 2.42 2.21(20:4n-3) 0.02 0.04 0.84 0.37 0.56 0.84 0.8920:5n-3 0.11 0.20 13.15 7.20 6.65 14.53 14.6322:1 0.00 0.01 0.00 0.18 0.00 0.61 0.5822:4n-6 0.03 trace 0.00 0.00 0.00 0.00 0.0022:6n-3 0.31 0.54 8.31 6.46 6.95 9.66 12.2424:0 0.00 0.00 3.14 2.03 2.34 4.19 3.56

Ratio 20:5n-3/22:6n-3 0.37 0.38 1.58 1.11 0.96 1.50 1.20

Table 6b. Fatty acid content in 18-d-old turbot larvae (pig mg D W “ 1) from the different treatments. Iso = Isochrysis, Rho = Rhodom onas. (-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

14:0 0.94 1.55 0.92 1.2114:1 0.00 0.00 0.00 0.0016:0 8.26 10.60 10.05 10.9616:1 2.32 2.88 2.42 2.9818:0 4.54 5.71 5.55 6.2218:1 18.21 20.75 21.68 22.0318:2 3.47 4.02 3.77 2.2218:3n-6 1.33 4.32 2.36 0.0018:3n-3 9.95 10.82 10.79 13.93(18:4) 1.44 1.45 0.00 2.3320:0 0.25 0.35 1.44 0.2920:1 1.15 1.20 0.29 1.26a 0.23 0.66 0.38 0.0020:2 0.52 0.69 1.40 0.7020:3n-6 0.22 0.19 0.67 0.2520:4n-6 1.00 1.34 0.27 1.6220:3n-3 1.48 1.63 1.92 2.07(20:4n-3) 0.68 0.65 0.81 0.0020:5n-3 8.66 9.52 10.26 13.7022:1 0.00 0.00 0.43 0.0022:4n-6 0.00 0.00 0.00 0.0022:6n-3 6.86 7.69 10.02 10.5124:0 2.49 2.19 0.00 3.43

Ratio 20:5n-3/22:6n-3 1.26 1.24 1.02 1.30

3). 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 o f 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 D una­liella, larval growth in the Pavlova tanks was similar to that in the R hodom onas 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

i c e s m ar. Sei. Symp., 201 (1995) Algae in the growth and survival o f turbot larvae 183

Table 7. Larval dry weight (D W ), survival rate, D H A and EPA content and E P A /D H A ratio on day 18 and D H A and E P A content in algal-enriched rotifers.

Algal species in larval tanks

Larval DW day18 (mg)

D H A in residual rotifers

D H A in larvae day 18

Larval survival

to day 18

EPA in residual rotifers

EPA in larvae day 18

E P A /D H Aresidualrotifers

E P A /D H A larvae day 18

Dunaliella 1 1.13 0.00 8.31 7.79 0.00 13.15 _ 1.58Dunaliella 2 1.01 1.32

Chlorella 1 0.77 0.47 6.46 42.28 1.04 7.20 2.22 1.11Chlorella 2 0.81 6.95 27.55 6.65 0.96

Pavlova 1 1.95 0.52 9.66 18.78 0.58 14.53 1.13 1.50Pavlova 2 2.05 12.24 24.39 14.63 1.20

Isochrysis 1 3.49 0.42 6.86 19.13 0.28 8.66 0.65 1.26Isochrysis 2 1.11 7.68 22.78 9.52 1.24

R hodom onas 1 2.01 0.62 10.02 38.15 0.49 10.26 0.97 1.02Rhodom onas 2 1.96 10.51 36.82 13.70 1.30

E

gQ

5

4

3

2

010 20 30 40 50

Survival (%) to day 18 from hatching

Figure 4. Turbot larval D W ( ± V v a r ) and survival to day 18 from hatching. Larvae were reared in intensive systems using static water and five different species o f monoalgal cultures. Dunaliella (circle), Chlorella (square), Pavlova (triangle up), Isochrysis (triangle down), and R hodom onas (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 Tetra- selmis 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 R hodo­

monas 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 100000 CFU ml-1 . The CFUs in the duplicates were similar and the differences be­tween treatments were less than 1 log, thus p er 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. algi- nolyticus 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

184 J. G. Støttrup, K. Gravningen, and N. H. Norsker

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 o f 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 (newly- hatched; 60 jjun) to interest the fish larvae, which by then were feeding on the much larger Artem ia 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 o f the observed effects

Næss et al. (1990) argued that the effect o f 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 o f certain free amino acids acting as feeding stimulants has not been

ICES mar. Sei. Symp., 201 (1995)

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 o f 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 o f 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 o f 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 a l. , 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 Aero-

monas 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 a l., 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

ICES mar. Sei. Symp., 201 (1995) Algae in the growth and survival o f turbot larvae 185

use of antibiotics is widespread in commercial hatcheries

for marine fish and shellfish (Nicolas et a l. , 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.

Acknowledgements

Thanks to the staff at Tinfos Aqua A/S, Norway, for

their invaluable help with the different cultures and the numerous sampling and to Dr Joachim Stoss for his encouragement and suggestions: Torben Kristensen, Danish Institute for Fisheries, Technology and Aqua­culture, Denmark, for his help and assistance with the gas chromatograph and Dr H. J. Fyhn, Bergen Univer­

sity, Norway, for the analyses of amino acids.

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