Nutritional value of diets that vary in fatty acid composition for larval Pacific oysters (Crassostrea gigas)

Aquaculture 143 (19%) 379-391

Nutritional value of diets that vary in fatty acid composition for larval Pacific oysters ( Crassostrea gigas) Peter A. Thompson

a’*, Ming-xin Guo b, Paul J. Harrison ’

a CSIRO Marine Laboratory, P.O. Box 20, North Beach, WA 6020. Aiutralia b Deparrment of Oceanography, University of British Columbia, Vancouver. B.C. V6T 124, Canada ’ Departments of Botany and Oceanography, Universiry of British Columbia, Vancouver, B.C. V6T 124, Canada Accepted 8 February 19%

Abstract High-light grown cells of Thalassiosira pseudonana and Pavlova lutheri constituted a superior diet for larval Pacific oysters (C’rassostrea gigas) relative to the same phytoplankton species grown under low-light. Growth rates were higher and mortality was lower for those larvae fed high-light (HL) versus low-light (LL) grown phytoplankton cells of the same species. Based upon larval growth rates, these diets could be ranked: HL T. pseudonana > LL T. pseudonana = HL Pavlova lutheri > LL Pavlova lutheri. Diets consisting of T. pseudonma and Pavlova lutheri cells grown at HL and LL were similar in carbon, nitrogen, protein and carbohydrate, but not lipid content. Significant differences also existed in the fatty acid composition of the diets. Diets consisting of high-light grown T. pseudonana provided the lowest proportion of the essential fatty acid 20:5n-3 and the highest proportion of the fatty acid 16:O. Larval growth rate was negatively correlated with the proportion of the essential fatty acid 205~3 in their diet. Larval mortality was positively correlated with the proportions of dietary 16:O. Examination of data collected over a number of years suggests that an abundance of dietary 205~3 has a strong negative effect on larval growth rates. Keywords: Crassosrrea gigas; Fatty acids; Light; Lipids;

Phytopl&ton

* Corresponding author. Fax: 61-9-246-8233; e-mail: [email protected]. 0044-8486/%/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SOO44-8486(96)0 1277-X

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1. Introduction

Phytoplankton can vary in nutritional value to a variety of planktivorous animals (Davis and Guillard, 1958; Watanabe et al., 1983). During early experimentation in oyster aquaculture, variation in nutritional value of phytoplankton did not appear to be related to their general biochemical composition (Walne, 1963). These early findings suggesting palatability and digestibility were significant factors in determining the nutritional value of phytoplankton. Subsequent use of artificial diets and the availability of more sophisticated analytical techniques provided superior tools for investigating amino acid balance and fatty acid (FA) composition, which led to the discovery that several fatty acids were essential for larval oyster growth (Langdon and Waldock, 1981). Artificial diets continue to improve, yet they remain generally suitable only as supplements for live food in raising larval bivalves (Jones et al., 1984; Chu et al., 1987). Therefore, most hatcheries rely heavily upon live phytoplankton as a food source. Thus interest in screening new phytoplankton species, testing different mixtures of phytoplankton species and adjusting mixes during larval development to produce a greater yield of animals has been strong. One method of selecting a new phytoplankton species for testing has been to select those with an abundance of known essential FAs. Studies of phytoplankton physiology have demonstrated that FA composition is a phenotypically plastic property of each species which changes rapidly in response to light (Shifrin and Chisholm, 1981) and nutrient deprivation (Harrison et al., 1990). This phenotypic plasticity makes it possible for the biochemical composition within one species of phytoplankton to vary significantly depending upon the culture conditions. Thus, one can provide diets to planktivores that are different in their biochemical composition, but quite similar in digestibility (cell wall composition) and palatability. In this paper we report on experiments where larval Crussostreu gigas were fed two species of phytoplankton, each grown under high and low light. In previous research we have shown that phytoplankton cells grown under high light typically have more short-chain saturated FA such as 160 and less of the essential fatty acid (EFA) 20:5n-3 (Thompson et al., 1990). Contrary to expectation, those animals receiving diets high in 20:5n-3 grew more slowly and suffered greater mortality than those fed diets low in 20:5n-3. In this new research we test Thalassiosira pseudonana and Pavlova lutheri, a phytoplankton species high in the EFAs 20:5n-3 and 22:6n-3 (Volkman et al., 1989). 2. Materials and methods 2.1. Algal culture and medium T. pseudonana (Hustedt) Hasle and Heimdal (NEPCC No. 58), and Pavlova lutheri Droop (NEPCC No. 5) were obtained from the Northeast Pacific Culture Collection (NEPCC), Department of Oceanography, University of British Columbia. Cultures were grown in enriched natural seawater using the nutrient-enrichment solutions (ES) of Harrison et al. (1980). The medium was modified as in Thompson et al. (1991) and the procedures for making the medium are in Thompson and Harrison (1992) and Thompson et al. (1993). The 8-l phytoplankton cultures were grown in four computer-con-

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trolled turbidostats similar in design to those used by Plstgaard et al. (1987). High-light cultures were continuously illuminated by four Vita-lite fluorescent tubes, and low-light cultures by one tube. The following irradiances were measured using a Biospherical 4n meter at the center of the culture at normal operating cell densities: saturating white-light culture (HL), 140 pmol photons m-* s- ‘; low white-light culture (LL>, 11 ymol photons m-* s-i. Phytoplankton cultures were grown at 22°C. Cell densities were maintained at about (1.2-2.6) X lo9 cells l- I. Inflow nutrient concentrations were never limiting and these cells grew at the maximum rates for the prevailing conditions of light and temperature. The cultures were stirred at 60 rev min-’ with a 7.6 cm Teflon-coated magnetic bar, and bubbled with a mixture of air and CO,. The pH of most cultures was checked daily (range 8.2-8.5) and adjusted to 8.2 by varying the ratio of CO, to air. Determination of phytoplankton biomass in each culture was made once per day by measuring in vivo fluorescence with a Turner Designs Model 10 fluorometer, and cell counts utilizing a Coulter Counter Model TAll equipped with a population accessory. 2.2. Biochemical

composition

of algae and C .gigas

Samples for the determination of phytoplankton biochemical composition were collected every 3 days. Data from each phytoplankton culture over the duration of the experiment were pooled to provide a mean for each biochemical parameter for each treatment (algal diet). Particulate organic carbon and nitrogen (POC and PON) subsamples (25 ml) were collected on precombusted 13 mm Gelman A/E glass-fiber filters (nominal pore size 1 Frn) and were analyzed on a Carlo Erba CNS analyzer. Triplicate subsamples (100 ml) for total lipid were extracted in chlorofotm:methanol:water (Bligh and Dyer, 1959) and analyzed by the lipid charring technique (Marsh and Weinstein, 19661, using tripalmitin as a standard. Total lipid also contained chlorophyll a. Triplicate subsamples for determination of total polymeric carbohydrates were collected from the methanol:water fraction of the total lipid extraction, hydrolyzed in sealed borosilicate glass tubes containing 3 ml of 1 N H,SO, for 20 h at 100°C and analyzed by the phenol-sulfuric acid technique of Dubios et al. (1956). Subsamples (50 ml) for total protein were collected on precombusted 25 mm GF/F filters and stored at - 20°C for later protein analysis by the modified Lowry technique (Lowry et al., 1951; Dortch et al., 1984). Phytoplankton subsamples (3-5 1) for fatty acid determinations were collected by centrifugation, freeze-dried, and sealed in precombusted glass bottles filled with nitrogen gas. Prior to analysis, samples were frozen at -20°C. Samples were saponified and methylated as described in Whyte (1988). An internal standard (21:O) was included with each weighed phytoplankton sample prior to saponification and methylation, allowing for the estimation of the absolute quantity of FAs per unit dry weight. FAs were analyzed on a Hewlett-Packard 5890A gas-liquid chromatograph fitted with a DB 3 50% Cyanopropyl (J&W Science, Folsom, CA, USA) capillary column and identified by comparison with standards in accord with Ackman (1986). 2.3. Oyster culture C. gigas larvae were grown following the guidelines provided by Breese and Malouf (1975). Unenriched natural seawater (collected from a minimum depth of 7 m, 28%0) for

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the oyster experiments was prefiltered, first through 25 pm, then through 1 p,m cartridge filters and finally through a Gelman A/E glass-fiber filter. One batch of recently fertilized oyster larvae from Coast Seafood (Quilcene, Washington), was thoroughly mixed and allocated into one of 12 flasks resulting in approximately 12700 larvae in 5 1 of water. Water was changed every 3 days. To change the water, larvae were gently strained onto 100 p,rn Nitex netting, concentrated into 250 ml, and mixed. A 10 ml subsample was removed for growth and survival measurements and the remainder was added to 5 1 of filtered natural seawater at 22°C. After each water change, all treatments (diets) received an equal volume of phytoplankton cells, starting at 4.4 X lo9 km3 and rising to 1.3 X 10” pm3 phytoplankton over the duration of the experiment. This density of phytoplankton was considered sufficient to saturate the requirements of the larvae. Overnight losses of phytoplankton due to grazing were rarely greater than 20% as estimated from in vivo fluorescence, and the solution was returned to the original density at 24 and 48 h. Every 72 h the water was changed and the oysters were provided with fresh phytoplankton. Antibiotics (50 p,g I-’ streptomycin and 30 kg l- ’ penicillin G) were also added at each water change to reduce the possibility of problems with bacterial contamination (Boume et al., 1989). Oysters were bubbled with air injected near the bottom of the flasks. Oxygen in the flasks remained at about 6.5 mg I-’ during the experiment. Experiments were terminated after 12 days. Oysters were measured from umbo to opposite edge using images projected from an inverted microscope onto a calibrated digitizing table. At least 50 individuals per replicate were examined to determine mean size every 3 days. Growth rates were calculated as the slope of the least-squares linear regression of the natural log of oyster size versus time. Similarly, to estimate mortality, an initial sample containing about 570000 individuals was examined, and towards the end of the experiments a sample of not less than 50 individuals was examined. The slope of a least-squares linear regression of the natural log of the percentage of dead oysters versus time was used to estimate mortality. For each of the four diets (treatments) there were three replicate flasks with an initial density of about 13 000 larvae randomly drawn from the same initial population. Data for larval growth rates, size and mortality were assessed by a two-way ANOVA. The biochemical composition of the larval diets was measured four times over the duration of the experiment and the data were assessed by one-way analysis of variance (ANOVA). Where necessary, multiple comparisons were made by the Student-Newman-Keuls’ pairwise procedure (Sokal and Rohlf, 1969). If the biochemical data failed to conform to standards for normality or homoscedasticity, we performed a Kruskal-Wallace one-way analysis of variance on ranks. Unless otherwise stipulated, probabilities less than or equal to 0.05 were considered significant.

3. Results 3.1. Oysters Oyster larvae fed high-light @IL) grown Thalmsiosiru pseudonanu grew from a mean size of 159 to 261 p,rn in 12 days at 22°C. Larval oysters fed HL T. pseudonunu

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383

300

so

270

270

240

240

.Nm

1

150

12

9

6

3

0

Time (d)

HL Tp

LL Tp

HL PI

LL PI

Fig. 1. Growth and survival of larval C. gigas. (A) Increases in mean size over time for larval C. gigas fed phytoplankton cells grown under different conditions. Oyster larvae were fed HL grown T. pseudomma (T 1, LL T. pseuabnana (A), HL Paulova lutheri ( n ) or LL Pauloua lutheri (0). Each point represents the mean value from measurements of more than 100 larvae. Error bars are f 1 SD. Specific growth rates were calculated as least squates regression (line) of In size versus time for each diet over the period of larval age from 0 to 12 days. (B) Percentage of dead animals at day 12 when fed high-light (HL) grown T. pseudonana, low-light (LL) grown T. pseudonana, HL grown Paulova lutheri or LL grown Paulova lutheri. Error bars are +1 SD.

showed no lag phase in growth with shell size increasing exponentially during the 12 day period (Fig. l(A)). The oyster larvae fed a diet consisting of LL grown T. pseudonana showed slower initial growth and mean size was significantly smaller at day 12 than those fed I-IL T. pseudonana. Oyster larvae fed HL grown Puvlovu lufheri initially had high growth rates but slowed noticeably at day 6 and finished at day 12 not significantly different in size from those fed LL grown T. pseudonunu (Fig. l(A)). After 12 days, oyster larvae fed I-IL Puvlovu lutheri were 20% larger than those fed LL Puvlovu lutheri. Larvae fed LL grown Puvlovu lutheri grew poorly throughout the experiment. At the end of 12 days their mean size was only marginally larger than at the beginning of the experiment. In general, larval oysters fed either HL or LL grown Puvlovu lutheri showed signs of failing to increase in size exponentially towards the end of the experiment (Fig. l(A)). Growth rates were significantly different between

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Table 1 Growth and mortality hiah or low light

143 (1996) 379-391

of larval oysters fed diets of Thalassiosira pseudonana or Pavlova lutheri grown under T. pseudonana

Pavlova lutheri

Mean

Growth rate (dny - ‘) High light Low light Mean ( f SEM)

0.043 0.022 0.033A

0.021 0.005 0.013B

0.032X 0.014Y (0.0008)

Mortality f% day- ‘) High light Low light Mean ( + SEM)

2.15 8.65 5.70A

2.75 5.36 4.05B

2.75x 7.01Y (0.333)

Within respective

main effect comparisons,

means followed

by dissimilar

letters differ (P < 0.05).

treatments, with insignificant interaction effects (Table 1). Diets consisting of HL grown T. pseudonana produced the highest larval growth rates (Table 1). Diets of LL T. pseudonana and HL Puulovu lutheri did not produce significantly different growth rates, while those larvae receiving a diet of LL Puvloua lutheri had the lowest growth rates. The proportion of dead animals did not always increase steadily throughout the experiment, therefore mortality rates calculated over the duration of the experiment were quite variable (Table 1). However, those larvae fed HL grown T. pseudonunu had the lowest mortality, significantly lower than those fed on LL T. pseudonunu and LL Puvlova lutheri. Larvae fed diets of HL grown T. pseudonuna and HL grown Puvlovu lutheri had similar mortality rates of about 2.8% day-‘. These results are reflected in the proportions of dead larvae on day 12, which, from greatest to least, were those fed LL grown T. pseudonana > LL grown Puvlouu lurheri > HL Puvlovu lutheri = HL T. pseudonanu (Fig. l(B)).

Table 2 The biochemical composition of diets fed to larval Crassostrea gigas; high-light and low-light grown Pavlova lutheri, and high-light and low-light grown Thalassiosira pseua’onana. Values reported here are means f 1 SD Biochemical

composition

of algal diets (pg per cell)

Cell volume

(pm31 Carbon (n=4)

Nitrogen (n=4)

Protein (n=4)

Lipid (II=41

Carbohydrate (n=4)

(n = 11)

Thalassiosira pseuabnana High light Low light

7.6f 8.3f

1.1*0.2 1.4kO.2

4.3 f 0.8 4.3 + 0.8

3.5 f 0.5A 4.5 f 0.5B

1.2rto.9 1.2* 1.1

44.5 f 3.9A 42.6 f 7.4A

Pavlova lutheri High light Low light

9.5 f 3.6 7.0f 1.5

1.2kO.3 1.1 hO.2

3.6f 3.3f

6.1 k2.OB 5.1 f0.9B

2.2rt 1.5 0.9 f 0.2

38.2 f 9.3A 28.1 f 3.6B

1.2 1.3

Means within the same column followed

by different

1.0 1.0

letters are significantly

different (P < 0.05).

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3.2. Biochemical

composition

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385

of the larval diets

Diets consisting of Thalassiosira pseuabnana and Pavlova lutheri were very similar in general biochemical composition (Table 2). No significant differences were noted in carbon, nitrogen, protein, or carbohydrate per cell, although the intra-sample variability was quite high, which resulted in low power for the statistical analysis. The diet consisting of HL grown T. pseudonana had significantly less total lipid per cell than diets of LL T. pseudonana and HL and LL Pavlova lutheri (Table 2). Diets of LL

Table 3 Fatty acid composition

(% of total) for Thalassiosira pseudonana

and Pavlova lutheri grown under different

irradiauces Fatty acid

High light

lo:0 120 140 150 16:O 16:ln-7 16:ln-5 16:2n-7 16:2n-4 16:3n-4 16:4n-3 16:4n-1 18:O 18:ln-9 18: 1n-7 18:2n-6 18:2n-4 18:3n-6 18:3n-3 18:4n-3 20:2n-6 20:4n-6 20:3n-3 20:5n-3 22:4n-6 22:5n-3 22:6n-3 24: 1n-9

Pavlova lutheri

Thalassiosira pseudonana Low light

High light

Low light

Mean

SD(n=4)

Mean

SD(n=4)

Mean

SD(n=4)

Mean

SD(n=4)

0.19 BLD 8.20 0.68 21.5A 22.4A 0.32 1.94A 2.6OA 8.13A 0.28 0.52 0.57 0.19 0.28A 0.46A BLD 1.02 0.34A 5.22 BLD BLDA BLD 16.4A BLD BLD 5.25A 0.49

0.05 BLD 0.38 0.04 4.27 3.56 0.04 0.28 0.54 1.66 0.08 0.14 0.01 0.01 0.04 0.26 BLD 0.78 0.14 0.79 BLD BLD BLD 4.44 BLD BLD 0.45 0.35

0.18 BLD 8.51 0.68 11.7B 30.3B 0.44 3.02B 4.20B 3.59B 0.52 0.31 0.38 0.26 0.39B 0.35A BLD 0.24 0.36A 3.60 BLD BLDA BLD 20.9AB BLD BLD 4.83A 0.32

0.02 BLD 2.11 0.04 0.45 1.19 0.07 0.33 0.14 0.66 0.09 0.10 0.05 0.06 0.07 0.14 BLD 0.05 0.02 0.25 BLD BLD BLD 0.50 BLD BLD 0.17 0.04

0.13 0.12 10.2 0.20 18.2A 16.OC 0.24 0.13c O&C 0.25C 0.18 BLD 0.55 0.90 1.23C 2.56B 0.14 1.13 1.20B 6.70 0.37 0.54B 0.14 22.9B 1.21 0.20 9.36B 0.49

0.02 0.03 0.24 0.04 1.52 0.61 0.02 0.01 0.05 0.07 0.01 BLD 0.08 0.08 0.24 0.44 0.09 0.56 0.26 1.44 0.03 0.16 BLD 1.38 0.20 0.10 1.19 BLD

0.13 0.14 9.93 0.26 16.6A 14.lD 0.19 0.28C 0.77D 0.43c 0.33 BLD 0.43 0.58 1.68C 1.87C 0.24 1.17 1.89C 6.76 0.42 l.lOC 0.22 24.7B 1.40 0.32 8.OOB 0.54

0.02 0.03 1.90 0.03 3.15 0.43 0.02 0.18 0.14 0.34 0.05 BLD 0.09 0.15 0.44 0.53 0.15 0.59 0.29 2.77 0.06 0.38 0.10 4.48 0.16 BLD 0.37 0.17

Means within the same row followed by dissimilar letters differ (one-way ANOVA, Student-Newman-Keuls’ pairwise multiple comparisons procedure, P < 0.05). Statistical analyses were not conducted on fatty acids less than 1% of total. BLD, below level of detection, treated as zero for statistical purposes.

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grown T. pseudonana also had less total lipid per cell than those of HL and LL grown Pavlova lutheri. Compared with a diet of LL grown T. pseudonana, a diet of HL grown T. pseudonana contained significantly greater amounts (mg g-’ dry weight (DW)) of 160 and 16:3n-4, and significantly less of 16:ln-7, 16:2n-4, 16:2n-7, 16:4n-3, 18:ln-7, and 20:5n-3 (ANOVA, data not shown). Most of these differences were also statistically significant in the comparison of relative proportions of fatty acids (Table 3). A diet consisting of HL grown Pavlova lutheri contained significantly greater amounts (mg g-’ DW) of 16:0, 16:ln-7, 18:0, 18:2n-6, and less 16:2n-7 and 16:3n-4 than one of LL grown Pavlova lutheri (ANOVA, data not shown). These differences are also reasonably similar to those in the relative abundances of fatty acids (Table 3). Comparing the diet of HL T. pseudonana with the diet of HL Pavlova lutheri produced a large number of significant differences in FA composition (Table 3), and of the major FA (over 5% of the total) these included 16: 1 n-7, 16:3n-4 and 20:5n-3. Differences we detected in the biochemical composition of the diets provided to C. gigas larvae were limited to total lipid content of the phytoplankton cells and to their FA content. Although the differences were not always statistically significant, diets consisting of HL versus LL grown cells of both species had greater amounts of the major FAs 16:0 and 22:6n-3 and less of 20:5n-3.

4. Discussion There should be no doubt that, for most phytoplankton species, biochemical composition is affected, by irradiance. The widespread relationship between it-radiance and chlorophyll a content is one good example (Geider, 1987). Other work has demonstrated a link between n-radiance and fatty acid composition (Cohen et al., 1988; Sukenik et al., 1989; Thompson et al., 1990). Greater irradiance can result in increased proportions of short-chain saturated (140, 16:0) fatty acids (Orcutt and Patterson, 1974; Thompson et al., 1990), presumably in response to the abundance of reducing power produced by these phytoplankton. Conversely, polyunsaturated fatty acids (PUFA) such as 20:5n-3 are predominantly found in monogalactyl diglycerides (Opute, 1974) associated with membranes (Fuller and Nes, 1987). Some species increase these PUFAs under conditions of low irradiance, possibly as a result of increased chloroplast and thylakoid membranes (Kates and Volcani, 1966; Thompson et al., 1990). The data presented here suggest a similar link between irradiance and fatty acid composition for T. pseudonana and PavZova lutheri. However, it is worth noting that the biochemical composition of the phytoplankton was measured from continuous cultures where the requirements for independent replication were not met. Thus, to be statistically rigorous, we cannot argue a cause and effect for the differences in the biochemical composition of the phytoplankton; we can merely state whether the diets provided to the larval oysters were significantly different. Both T. pseuabnana and Pavlova lutheri cultures grown in high-light constituted a superior diet, as demonstrated by greater growth and reduced mortality, for C. gigas larvae relative to diets consisting of the same species grown in low light. As with any

PA. Thompson et al./Aquaculture .05 .

_

143 (1996) 379-391

I

/

I

A

I

.04 -

387

‘i. .03 -

d 0 S S

ik..

.02 -

g

.oi 0.00

*

-

I

$.I.,.

I

I

I

15

18

21

24

20:5n-3

in diet (% of total fatty acids)

27

90 g

a0

3

70

g 0 g

60

c

40

5

30

H

20

50

10 12

15

16

21

24

l&O in diet (% of total fatty acids)

Fig. 2. Growth rates and mortality for C. gigas fed either T. pseudomma or Paulooa lutheri grown under high or low light. (A) Growth rate versus proportion of the fatty acid 20:5n-3 in diet (n = 4, r2 = 0.99, P < 0.01). (B) Mortality versus the proportion of 16:O in diet (n = 4, r* = 0.92, P < 0.05). Oyster larvae were fed HL grown T. pseudonanu ( v 1. LL T. pseudonana (A ), HL Pavlova lutheri ( n 1, or LL Pauloua lutheri (0). Error bars are f 1 SD.

study which uses live phytoplankton, conclusions based upon these observations must be tempered by the possibility of effects due to variation in other unmeasured biochemical constituents. We believe, however, that the observation of inter-specific changes in nutritional value associated with irradiance provides some evidence that palatability and digestibility caused by the cell wall are unlikely to be confounding the results. If the nutritional value of phytoplankton cells was associated with FA composition it would be reasonable to expect an overall relationship between FA composition and nutritional value. If FA composition were the major factor, then this relationship might span species and experiments. From the experimental data presented here, we can demonstrate that the proportion of 205~3 in the larval diet was negatively correlated with the growth rates of the C. gigas larvae (Fig. 2(A)). Conversely the proportion of 16:0 in the larval diets was positively correlated with higher survival (Fig. 2(B)). Our examination of all the data we have collected over a series of experiments involving numerous batches of larvae and six different phytoplankton species continues

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389

species, larval growth was a highly significant negative linear function of the abundance of dietary 20:5n-3 (Fig. 3(B)). The overall negative correlation between dietary content of 20:5n-3 and larval growth rate must be considered tentative as it is based on experiments conducted over a number of years under slightly different conditions. In spite of this, we believe the results are important enough to merit consideration. They are very similar to those obtained by Dickey-Collas and Geffen (1992) for plaice larvae, which imply that, while small amounts of these FAs may be essential, large amounts may be deleterious. The phytoplankter Pavlova lufheri is a good example of those species that might be selected for aquaculture on the premise that a high abundance of the EFAs 20:.5n-3 and 22:6n-3 will result in nutritional superiority (Volkman et al., 1989). Some previous research has reported that Pavlova lufheri (formerly Monochrysis lutheri) was a good food item for bivalve larvae (Davis and Guillard, 1958; Walne, 1963), although more recent work has shown it to be a poor food for larvae of the Pacific oyster (Crassostrea gigas; Langdon and Waldock, 198 1) and the Japanese scallop (Patinopectin yessoensis; Thompson et al., 1994). In the present study, I-IL grown Pavlova lutheri was a markedly inferior food item relative to I-IL grown T. pseudonana and a superior food item relative to LL grown Pavlova lutheri for C. gigas larvae. Within this experiment, the higher larval growth rates were associated with less 20:5n-3 and greater amounts of 16:0. For most marine phytoplankton, the dominant saturated FAs are 140 and 16:0 (Ackman et al., 1968; Volkman et al., 1989), which provide the basic components of neutral lipids found in C. gigas larvae (Chu and Webb, 1984). It is known that greater amounts of neutral lipid in oyster larvae are associated with increased larval vigour, growth and survival (Gallager et al., 1986). In experiments with larvae of the Sydney rock oyster (Saccostrea commercialis), diets supplemented with cod liver oil, rich in FA 14:0 and 16:0, yielded by far the best growth rates and a higher survival (Numaguchi and Nell, 1991). In earlier work, we demonstrated a link between the proportion of saturated FAs in phytoplankton and the proportion of these saturated FAs in the oyster larvae (Thompson et al., 1993). It was suggested that the lack of a consistent relationship between dietary saturated FA content and larval growth rate could be due to differences between experiments. However, these present data indicated that within a single experiment there was no consistency in the relationship between the saturated FA content of different phytoplankton species and growth of larval C. gigas (Fig. 3(A)). While we still interpret the results to suggest that greater amounts of dietary saturated FAs may be beneficial, they increasingly suggest the possible deleterious effects of diets high in PUFAs (Fig. 3(B)). In conclusion, high-light grown phytoplankton cells were a nutritionally superior diet for C. gigas relative to low-light grown cells of the same species. The nutritional superiority of the phytoplankton cells was correlated with a decrease in the proportion of the polyunsaturated fatty acid 20:5n-3. Many phytoplankton species adjust FA composition in response to irradiance, temperature and nutrient status providing a number of methods whereby bivalve hatcheries may manipulate the nutritional value of their algal foods. Further research is needed to assess the ecological significance of variation in the FA composition of phytoplankton.

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Acknowledgements

The authors wish to acknowledge the assistance of N. Ginther and T. Larson and to thank Drs. J.N.C. Whyte, T.R. Parsons and Ljerka Kunst for the use of their equipment. Funds were provided by Operating and Strategic grants from the Natural Sciences and Engineering Research Council of Canada.

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