1988, Bolla 1989, Rigby 1990, Tocher and Mackinlay. 1990). Initial investigations highlighted the role of. 20 : 5 co 3 and 22 : 6 co 3 for good survival and growth ...
Marine Biology 113, 463 468 (1992)
Marine ==,.BiOlogy 9 Springer-Verlag 1992
Importance of the fatty acids 20:5o 3 and 22:6w3 in the diet of plaice (Pleuronectes platessa) larvae M. Dickey-Collas and A. J. Geffen University of Liverpool, Port Erin Marine Laboratory, Port Erin, Isle of Man, Great Britain Date of final manuscript acceptance: February 21, 1992. Communicated by J. Mauchline, Oban
Abstract. The requirement of plaice (Pleuronectes platessa L.) larvae for the fatty acids 20:5co3 and 22:6(o3 was studied. Larvae were reared from first feeding to beyond metamorphosis on Artemia sp. nauplii (EG brand), whose nutritional content had been manipulated by enrichment. Some larvae were fed Artemia rich in 20:5 co 3 and 22:6 co 3, and others were fed nauplii with no 22:6 co 3 and low levels of 20:5 co 3 (1.9 % of the total fatty acids). The differences in diet had no significant difference on growth or survival of the plaice larvae. The results indicate that the plaice larvae in this study did not require 22:6 co 3 in their diet, even when the levels o f 20:5 co 3 were low.
1974). Few nutritional studies of plaice have recently been undertaken since the research emphasis has shifted to flatfish species of a higher marketable value. The present study investigated the importance of 20 : 5 co 3 and 22: 6 co 3 in the diet of larval plaice. Larvae were reared on newly hatched and on enriched Artemia sp. to determine if the presence of dietary 22: 6 co 3, at low levels of 20 : 5 co 3, was critical to the survival of plaice larvae. The Artemia used were 20: 5 co 3- and 22: 6 co 3-deficient and an enrichment was chosen that increased the levels of these fatty acids. Some larvae were also offered periods of newly hatched, and periods o f enriched, nauplii to investigate whether variations in the quality of the diet would affect the developing larvae.
Introduction
Materials and methods
The fatty acid requirements o f flatfish and flatfish larvae have been well studied, and four probable essential fatty acids have been identified; 18 : 2 co 6, 18 : 3 co 3, 20 : 5 co 3 and 22:6 co 3 (Owen et al. 1975, Gatesoupe et al. 1977, Leger et al. 1979, Bell et al. 1985, M i n k o f f 1987, Tzoumas 1988, Bolla 1989, Rigby 1990, Tocher and Mackinlay 1990). Initial investigations highlighted the role o f 20 : 5 co 3 and 22 : 6 co 3 for good survival and growth under cultured conditions (Bell et al, 1985, Peleteiro et al. 1988, Bolla 1989). These two fatty acids are m o r e abundant in natural prey than in cultured live foods, e.g. rotifers and Artemia sp. (Watanabe et al. 1983, Dendrinos and Thorpe 1987), and their importance to the larvae of certain flatfish m a y have been over-emphasised. Recent work has shown that turbot (Scophthalmus maximus L.), Dover sole (Solea solea L.) and plaice (Pleuronectes platessa L.), do not require 22: 6 co 3 in the diet if adequate 20:5 co 3 is present ( M i n k o f f 1987, Tzoumas 1988, Linares and Henderson 1991). However the m i n i m u m requirements o f plaice larvae for 20: 5 co 3, as a precursor for 22 : 6 co 3, has not yet been determined. Early flatfish experiments used plaice (Pleuronectes platessa) as a trial species (Bowers 1974, Shelbourne
Pleuronectes platessa L. larvae were fed either enriched (P1) or newly hatched (P4) Artemia sp. nauplii to metamorphosis, or were fed newly hatched nauplii throughout their development except for a 1 wk period (P2 and P3) when they received enriched nauplii (Table 1). The Artemia lEG brand, Artemia Systems (AS), Gent] were enriched with Super Selco (AS). This is a fatty liquid which is emulsified prior to addition to the Artemia tanks. Incubation, washing and enrichment were carried out as recommended by AS. The fish larvae were fed the newly hatched brine shrimp naupliijust after hatching (Instar I), whereas the enriched Artemia were 24 h old (Instar II), having been enriched for 12 h. A known number (I00000 to 250000) of nauplii were collected on a pre-dried, weighed Whatman glass-fibre filter (GF/A) and dried to constant weight at 100 ~ Their dry weight was determined to the nearest 1 mg. Larger samples used for further analysis were collected in a 150 gm mesh and freeze-dried for 72 h. Samples were stored in oxygen-free, sealed vials at -30~ for 2 wk. The relative proportion of protein was determined by the Kjeldahl method, using 6.25 times the percentage nitrogen to calculate the percentage protein. The lipids were extracted for 20 min at 4 ~ in 2 ml of 2:1 chloroform methanol (Folch etal. 1957). After centrifugation, 0.8 ml of chloroform was added, and 0.64 ml of distilled water, for further centrifugation. The lower phase was dried at 100~ for 30 min. When cool, 1 ml of chloroform was added and three 50 gl aliquots were taken for total lipid determination. After the addition
M. Dickey-Collas and A.J. Geffen: Essential fatty acids of plaice larvae
464
of 0.5 ml of concentrated H2SO4 the dried sample was charred at 250~ for 15 min (Marsh and Weinstein 1966, Holland and Hannant 1973). Once cool, 2.5 ml of distilled water were added and the absorbance read at 375 nm on a Philips PU 8670 spectrophotometer. The percentage lipid, by dry weight, was determined by comparison to a tripalmitin calibration curve. The level of ash was determined by ashing dried samples at 500~ for 5 h (Allen et al. 1974). The unaccounted proportion of the nauplii was assumed to be carbohydrate. The calorific content of the nauplii was determined by summing the calorific conversion values of the proportions of lipid, protein and carbohydrate (Jobling 1983). Analysis of the fatty acids of the brine shrimp was carried out using the methods of Rigby (1990), based on those of Folch et al. (1957). Extraction from the dried nauplii was carried out as for the lipid determination. The chloroform methanol mixture contained 0.01% butylated hydroxytoluene (BHT). The solids were removed from the solvents through a coarse filter (Whatman 1) and 0.2 vol of 0.017% aqueous MgC12 were added. After vigorous shaking, the emulsion formed was immediately centrifuged for 10 min. The lower phase was retained, washed with Folch upper-phase solvent (Folch et al. 1957) and passed through chloroform-soaked phaseseparating paper (Whatman 1PS). The chloroform was evaporated at 50 ~ with a Buchii Rotovaporator. The samples were methylated with 2.5% HzSO 4 in dry methanol at 70 ~ for 2 h. The solution was then neutralised with NaHCO 3 and NaC1 and the methyl esters were dissolved in petroleum ether. The petroleum ether was then evaporated under nitrogen and the sample redissolved in chloroform. The methyl esters of the fatty acids were separated by thinlayer chromatography, with 19:1 petroleum ether diethyl ether as the solvent. The fatty acid methyl esters were then stored in 500 I~1 of hexane, in oxygen-free sealed vials at -30 ~ for later gas-liquid chromatography (GLC) on a G.C. 6000 Carlo Erba Vega Series chromatograph with a 10 m x 0.32 mm i.d. fused silica column coated with polyethyleneglycol Superox 4, linked to an integrator. The programme used was as follows: after injection, the temperature was held at 80~ for 1 min, increased at 50~ to 135~ then increased at 4~ to 200~ at which temperature it was held for a further 15 min. The prominent peaks were all identified by comparison to standards and their relative concentrations are expressed as percentage of the total fatty acids.
The larvae were hatched from eggs obtained from adult plaice caught in the Irish Sea, southeast of the Isle of Man. Hand-stripped eggs from three females were mixed with milt of two males. Incubation and hatching took place at 10 ~ in 60-1itreblack conical tanks, filled with sterilized seawater and the water was gently aerated. Illumination was 10 to 13gmol photons m - 2 s -1 on a 16h light: 8 h dark cycle. Half of the volume of water was changed daily until hatching occurred. Day 1 was counted as the day when 50% hatch occurred, this was 12 d after fertilisation. The larvae were reared in 12 black, circular, 60-1itrePVC tanks, filled with sterilized seawater and initially stocked at 15/litre. Illumination was continuous at 4 lamol photons m 2 s- 1. The temperature was not controlled and ranged between 12 and 15~ with a mean of 12.8 ~ Light aeration was provided by airstones. Threequarters of the volume of water per tank was exchanged every other day with sterilized seawater. Dead larvae were siphoned daily from the tank bottom and counted. The experiment ran until the larvae were 50 d old; metamorphosis occurred around Day 35. All the larvae were fed with newly hatched Artemia sp. nauplii and algae-reared rotifers from Days 5 to 7. The four nutrition treatments (Table 1) commenced on Day 8 and ran to Day 50. Densities of the brine shrimp were maintained at 5/ml in all rearing tanks. Each treatment was replicated in three tanks. At Day 6 and then on Days 14 and 20, twently larvae were randomly sampled from each tank with a hand net, anaesthetised and their lengths determined with a dissecting microscope. At the end of the trial, all surviving metamorphosed fish were anaesthetised, measured with calipers and frozen for dry weight determinations. The growth and survival of the plaice larvae to Day 50 were compared by ANOVA (Minitab Statistical Package, Version 7). The growth data were tested for normality prior to analysis. Daily survival was calculated by the summation of the number of dead on each day to the final survivals of each tank. Before Day 8, the larvae decomposed too quickly for the daily mortality counts to be accurate, so this process was used calculating backward from the final day to Day 8. Differences in the dry weights of the pooled larvae and juveniles were tested with ANOVA. The biochemical differences in the brine shrimp nauplii were also compared by ANOVA of angular transformed data.
Results Table 1. Experimental design of Pleuronectes platessa rearing trial, showing feeding schedule of plaice larvae. Day 1 is taken as day of 50% hatch. Each treatment had three replicates. SS: plaice fed Super Selco-enriched Artemia nauplii; NH: plaice fed newly hatched, unenriched Artemia nauplii Experimental treatment P1 P2 P3 P4
Type of Artemia fed on Days: 8-15
16-23
24-31
32-38
39-50
SS SS NH NH
SS NH NH NH
SS NH SS NH
SS NH NH NH
NH NH NH NH
Although the weight and calorific content of the newly hatched Artemia sp. nauplii did not differ significantly from those of the enriched nauplii (Table 2), enrichment did change the proximate composition of the nauplii (Table 2, Fig. 1). Super Selco significantly increased the proportion of lipid in the brine shrimp (15.7 and 23.2% in newly hatched and enriched nauplii, respectively; ANOVA, F= 13.69; 5 dJ~p < 0.05), and resulted in significantly less protein (1.44 and 1.26 pg per nauplius, respectively; ANOVA, F= 151.3; 5 d~ p
