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The lipids of the cysts of freshwater- and marine-type Artemia
327
,4quaculture, 109 ( 1993) 327-336 Elsevier Science Publishers B.V., Amsterdam AQUA 50037
The lipids of the cysts of freshwater- and marinetype Artemia J.C. Navarrob, F. Amatb and J.R. SargenV “N.E.R. C. Unit ofAquatic Biochemistry, School of Natural Sciences, Stirling University, Stirling, UK blnstituto de Acuicultura de Torre de la Sal (C.S.I.C.), Ribera de Cabanes (Castekin), Spain (Accepted 6 May 1992)
ABSTRACT
Navarro, J.C., Amat, F. and Sargent, J.R., 1993. The lipids of the cysts of freshwater- and marine-type Artemia. Aquaculture, 109: 321-336. Cysts of marine-type Artemia had approximately twice the level of total lipid per unit dry weight as cysts of freshwater-type Artemiu. Total lipid of freshwater-type cysts had higher percentages of pigments, cholesterol and free fatty acids and lower percentages of triacylglycerols than total lipid of marine-type cysts. The percentage of phospholipids in total lipids was the same for both types of cysts. The phospholipids and triacylglycerols of marine-type cysts had substantial levels of 20: G-3 and 20: 4n-6 whereas freshwater-type cysts lacked these fatty acids but had elevated levels of I8 : 3n-3. The ratio 16: O/ 16 : 1 was higher in lipids, especially neutral lipids, of marine-type than of freshwater-type cysts. Lipids of both types of cysts had relatively low percentages of polyunsaturated fatty acids, with the fatty acid composition of the major individual phospholipids and neutral lipids being broadly similar. Artemia nauplii are the most widely used larval food in aquaculture, so the significance of the results for the culture of marine fish and crustacean larvae is discussed in relation to the importance of considering absolute rather than relative levels of polyunsaturated fatty acids in Artemiu and the phenotypic plasticity of the genus.
INTRODUCTION
Newly hatched nauplii of the brine shrimp, Artemia sp., are the most widely used larval food in aquaculture but their nutritional value is not completely satisfactory for marine fish and prawn larvae. All stocks of Artemia contain 18: 3~3, some but not all contain 20: 5~2-3,and none contains 22: 6n-3 in more than trace amounts. Watanabe et al. ( 1978a, 1980) classified Artemia into two general groups, freshwater-type hernia devoid of 20: 5n-3 and thus only suitable for feeding to freshwater animals, and marine-type Artemia containing 20: $2-3 and allegedly suitable for feeding to marine animals. This Correspondence to: Dr. J.C. Navarro, Instituto 12595 Ribera de Cabanes, Castellh, Spain.
00448486/93/$06.00
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de Torre de la Sal (C.S.I.C.),
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classification was based on the assumption that freshwater animals can convert 18 : 3n-3 to 20: 5n-3 and thence to 22 : 6n-3, and that marine animals cannot convert 18 : 3n-3 to 20: k-3 but can convert 20: h-3 to 22 : 6n-3. Since then, many studies have been devoted to characterising Artemiustocks from different geographical origins from the point of view of the fatty acid compositions of their lipid. High variability among strains and also among batches of the same strain has been reported. Nonetheless, when 18 : 3n-3 is present in high amounts, 20: h-3 is generally absent or present in very low amounts (Leger et al., 1986) in line with the freshwater-marine distinction described above. Consequently the distinction continues to be useful as a general guide, although for quantitative nutritional studies causes of variations in the quality of Artemia become important. Navarro et al. ( 1992 ) found no clear correlation between the polyunsaturated fatty acid (PUFA) composition of Artemia cysts and the coastal/inland location of their saline habitats. However, Lavens et al. ( 1989) and Navarro and Amat ( 1992) demonstrated that the diet of broodstock Artemia influenced the fatty acid composition of both cysts and newly hatched nauplii. Navarro et al. ( 1991) showed that approximately 80% of the total lipid of Artemia cysts was neutral lipid, which is strongly influenced by diet in most animals (Love, 1980; Gurr and Hat-wood, 199 1). Watanabe et al. ( 1978b), Sakamoto et al. ( 1982) and Leibowitz et al. ( 1987) have all reported fatty acid compositions of total neutral and total polar lipid fractions of Artemia nauplii. However, to our knowledge, fatty acid compositions of the individual phospholipids of Artemia cysts have not been described. We report here analysis of the levels and fatty acid compositions of neutral lipids and individual phospholipids in cysts from freshwater-type and marine-type Artemia and discuss the implications of the findings for aquaculture. MATERIALS AND METHODS
Cysts from the Artemia population of the “La Mata” coastal lagoon (Torrevieja, Alicante, Spain) harvested by the authors in July 1985 and belonging to a parthenogenetic diploid strain were selected as marine-type cysts (LMT ) . Cysts of commercial origin (A.T.P., S.A. ) known to be deficient in essential fatty acids for marine organisms were chosen as freshwater-type cysts (GSL) . The latter cysts were further analysed by electrophoretic techniques (Seidel et al., 1980; Requintina and Simpson, 1987) and were assessed as belonging to a population from Great Salt Lake (Utah, U.S.A.) (Navarro, 1990). Prior to lipid extraction, cyst samples were hydrated in seawater (salinity 34 g/l) until the cysts were observed to be spherical under a dissecting microscope. They were then decapsulated according to the procedures described in Bruggeman et al. ( 1979). After a thorough wash in distilled water, the cysts were blotted over soft paper tissue and divided into six aliquots, three of which
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CYSTS
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were dried for 24 h at 110’ C to calculate the moisture content, the remaining three being used for lipid extraction. Preliminary studies showed no differences between the lipid classes of decapsulated and whole cysts. Therefore, decapsulated cysts were analysed here because their lipids were easier to extract, Lipids were extracted using the method of Folch et al. ( 1957), quantified gravimetrically using an analytical balance (Mettler H 64) and stored at 20’ C at a concentration of 10 mg/ml in chloroform:methanol (2 : 1, V.-V)containing 0.0 1% ( WY) of butylated hydroxytoluene (BHT) as antioxidant. Before sealing the vials, nitrogen was flushed through the samples to create an anoxic atmosphere. Extractions were carried out in triplicate. Lipid classes were analysed quantitatively using double development, high performance thin layer chromatography (HPTLC ) on 10 x 10 cm plates of silica gel 60 (E. Merck, Darmstadt, Germany) as detailed by Olsen and Henderson ( 1989). Individual samples were analysed in quintuplicate with appropriate lipid standards and blanks. After development, the plates were charred with a copper acetate-phosphoric acid reagent (Fewster et al., 1969 ) and analysed by quantitative scanning densitometry (Olsen and Henderson, 1989). Individual lipid classes were separated on 20 x 20 cm plates of silica gel 60 (E. Merck, Darmstadt, Germany) using the polar solvent system of Olsen and Henderson ( 1989), detected by spraying with 0.1% 2’ ,7’-dichlorofluorescein and subjected to acid-catalysed transmethylation (Christie, 1982 ) after adding 19 : 0 as an internal standard. Fatty acid methyl esters were extracted from the transmethylation reaction with hexane:diethyl ether ( 1: 1, v:v) and purified on 20 x 20 cm silica gel G 60 plates using hexane:diethyl ether:acetic acid (85 : 15 : 1.5, VXV) as solvent. Fatty acid methyl esters were finally analysed by high resolution capillary gas-liquid chromatography as detailed previously (Navarro et al., 1992). Total lipids and lipid classes were analysed using a Student t test (Zar, 1984), at a 95% (PC 0.05) confidence level. Prior to statistical analyses, percentage compositions were arcsine transformed (Zar, 1984). RESULTS
AND DISCUSSION
Table 1 shows that cysts from the marine-type Artemia (LMT) had twice the total lipid per unit dry weight as the freshwater-type samples (GSL). Leger et al. ( 1986) reported that mean total lipid levels of Artemia nauplii accounted for 18.92 + 4.5% of the dry weight. Even though deviations can be expected, freshwater-type Artemia samples tend to have lower total lipid levels than the marine-type samples (Navarro et al., 1992). Moreover, lipid levels in cysts are known to be lower than in nauplii (Dutrieu, 1960; Schauer et al., 1980; Navarro et al., 1991). Nonetheless, the value of 9.2% found here
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TABLE 1 General lipid content of the cysts of freshwater (GSL)- and marine (LMT)-type Type of lipid
GSL
LMT
Total lipids (% dry weight) Neutral lipids/polar lipids n-3 HUFA (mg/g dry weight ) Fatty acids (mg/g dry weight)
9.2 f 0.2a 6.5 0.3 54.8 + 4.0a
17.9+0.4b 6.4 10.2 157.6f2.0b
Artemia cysts’.’
‘Data are means k standard deviations of triplicates, except for the ratio neutral/polar and n-3 HUFA which were calculated directly from means in Tables 2, 3 and 4. *Within the same row, different letters denote significant differences (PcO.05). When no letters are present statistical analyses were not performed. TABLE 2 Lipid classes in freshwater (GSL)- and marine (LMT)-type Lipid class
I of total lipids GSL
Polar Sphingomyelin/ lysophosphatidylcholine Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid/ cardiolipin Phosphatidylethanolamine Neutral Pigments Cholesterol Free fatty acids Triacylglycerols Cholesterol esters Polar lipids Neutral lipids
decapsulated Artemia cysts’ mg/g dry weight
LMT
GSL
LMT
0.5 i 0.2a
0.4+0.la
0.7 f O.Oa
0.7kO.la
7.3kO.la 0.2kO.la 1.6kO.Oa
7.5+0.6a 0.3kO.Oa 1.5kO.la
7.2fO.Oa 0.2kO.la 1.5If:O.Oa
13.52 I.lb 0.5kO.Ob 2.8+0.2b
0.5kO.la 2.220.3a
0.7+O.Ob 3.2kO.lb
0.6 + O.Oa 2.2+0.3a
1.3+0.lb 5.8+0.lb
32.4f l.Oa 12.5f0.2a 9.6 f0.5a 23.4+ 0.2a 9.7+0.7a
12.2k0.7b 9.2 ? 0.4b 5.4+ 1.5b 51.9+2.3b 7.8f 1.3a
26.2+0.1a 12.420.2a 9.7+0.5a 22.9 + 0.2a 9.9 + 0.8a
19.3+ 1.3b 16.8k0.7b 10.7k2.8a 95.7f3.9b 15.1 f 2.7b
12.7 k 0.7a 87.6+0.6a
13.6f l.Oa 86.6* l.Oa
12.510.5a Sl.Ot0.7a
24.7 k 1.3b 157.5?2.7b
‘Means in the same row and unit with different letters are significantly different (a=0.05).
for the total lipid in the freshwater-type GSL cysts is lower than anticipated. The lipid level in the marine-type LMT sample coincides with the mean value reported in the literature (LCger et al., 1986 ). Table 1 shows, from calculations using the lipid class compositional data in Table 2, that the ratio of polar/neutral lipids of the marine-type LMT and the freshwater-type GSL remain unchanged despite the the former having greater amounts of both neutral and polar lipids (Table 2 ) . Likewise Table 1
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CYSTS
331
shows, from calculations using the fatty acid compositional data of total lipid in Tables 3 and 4, (a) a substantially higher level of total fatty acids per unit dry weight in marine-type (LMT) as compared to freshwater-type (GSL) cysts; and (b) higher levels of n-3 highly unsaturated fatty acids (HUFA), i.e. n-3 PUFA of chain lengths > C20, in the marine-type (LMT) as compared to the freshwater-type (GSL) cysts. Therefore, on the basis of the nutritional provision of fatty acids, in general, and HUFA, in particular, to marine fish and crustacean larvae, the freshwater-type cysts are much inferior to marine-type cysts. This stems from their having a lower percentage of glycerides in total lipid, a lower percentage of n-3 HUFA in glycerides, and also a lower content of total lipid per unit weight, as compared to marine-type cysts. This illustrates the importance in larval nutrition of expressing HUFA levels in live foods on an absolute basis, e.g. mg of HUFA per g of dry weight, rather than simply on the basis of percent fatty acid compositions. Table 2 shows that some 13% of the total lipid of both freshwater- and marine-type cysts was present as polar lipids. Phosphatidylcholine (PC) followed by phosphatidylethanolamine (PE) were the major polar lipids present and, although some differences occurred in percentage compositions of individual polar lipids between freshwater-type and marine-type cysts, e.g. PE had a higher percentage in marine-type LMT than in freshwater-type GSL, the differences were not marked. The situation for neutral lipids, which accounted for 87% of the total lipid in both GSL and LMT, is different. Thus, the marine-type LMT had twice the percentage of triacylglycerols (TAG) in total lipids as the freshwater-type GSL. In contrast, GSL had a markedly higher percentage of pigments and significantly higher percentages of both cholesterol and free fatty acids than LMT. That is, the total lipid of the freshwatertype GSL had a higher percentage of non-saponifiable lipid and conversely a lower percentage of fatty acids than that of the marine-type LMT. In terms of providing fatty acids, the lipid of GSL is clearly inferior to that of LMT. However, when the data in Table 2 are expressed as per g dry weight of cysts, so taking account of the different absolute lipid levels noted in Table 1, the amounts of pigments, cholesterol and free fatty acids are more similar in the two types of cysts (although significant differences do occur in the levels of pigments and cholesterol). At the same time, the levels of individual phospholipids, especially PC and PE, and TAG are markedly elevated in the marine-type LMT as compared to the freshwater-type GSL. The data for the fatty acid compositions of total lipid from marine-type (Table 3 ) and freshwater-type (Table 4 ) cysts conform to earlier analyses for Artemia total lipid (Watanabe et al., 1978a,b, 1983; LRger et al., 1986; Navarro, 1990). Thus, the freshwater-type (GSL, Table 4) had 18 : 3n-3, 18 : 2n6 and some 18 : 4n-3 as major PUFA but only traces of C20 PUFA. In contrast, the marine-type (LMT, Table 3) had 20: 5n-3 and 20: 4n-6 as well as 18 : 3n-3, I8 : 2n-6 and 18 : 4n-3 as major PUFA. Tables 3 and 4, however, ex-
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ET AL.
TABLE 3 Fatty acid composition Mata’
of the lipid classes and total lipids of the marine-type Arfemia cysts from La
Fatty acid
Lipid class (O/oof total fatty acids)
14:o 15:o 16:0 16: ln-9 16: In-7 16:2 16:3 18:O 18: In-9 18: In-7 18:2n-6 18:3n-3 18:4n-3 20:o 20: In-9 20:4n-6
20:4n-3 20: 5n-3 22:ln-I1 22: 5n-3 Identified peaks n-3 HUFA n-6 HUFA n-3 PUFA n-6 PUFA n-3/n-6 Saturated Monoenes PUFA
PC
PS
PI
PE
TNL
TL
0.6 0.8 13.3 1.7 6.8 1.4 1.7 5.8 28.4 8.4 3.9 1.7 I.1 nd nd 1.3 nd 5.4 nd nd 82.3 5.4 1.3 8.2 5.2 1.6 20.5 45.3 16.5
2.6 2.6 15.3 3.2 3.8 1.7 1.6 9.3 21.8 5.3 2.9 4.1 nd 0.5 1.1 nd nd 2.1 0.6 1.1 79.7 3.3 0 7.4 2.9 2.6 30.3 35.8 13.6
1.8 2.2 11.8 2.5 3.7 1.8 1.4 10.5 21.9 9.5 3.4 3.6 nd nd 1.4 2.1 nd 4.6 nd 1.0 83.2 5.6 2.1 9.2 5.5 1.7 26.3 39.0 16.9
0.9 1.5 8.9 ns 7.7 1.7 1.6 5.7 33.6 11.3 4.1 2.8 nd nd 0.9 2.3 nd 5.4 nd nd 88.4 5.4 2.3 8.2 6.4 1.3 17.0 53.5 17.9
1.7 0.8 15.2 1.8 12.2 1.4 2.4 3.0 26.8 11.6 4.5 2.6 1.1 nd 0.7 0.9 0.3 5.2 nd nd 92.2 5.5 0.9 9.2 5.4 1.7 20.7 53.1 18.4
1.3 0.6 13.8 ns 12.5 1.5 2.5 3.5 24.7 10.0 4.7 2.7 1.3 nd 0.5 1.2 tr 6.5 0.1 nd 87.4 6.5 1.2 10.5 5.9 1.8 19.2 47.8 20.4
Ins, not separated from the other monoene; nd, not detected; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; TNL, total neutral lipids; TL. total lipids.
tend previous analyses by documenting the fatty acid analyses of individual phospholipids. All of the phospholipids listed in Table 2 were analysed but, for the sake of clarity and because no obviously notable features were present, the data for the “unresolved pairs” sphingomyelin + lysophosphatidylcholine and phosphatidic acid + cardiolipin are omitted. The fatty acid compositions of total lipid and total neutral lipid were similar in the marine-type LMT (Table 3). This is to be expected since neutral lipid comprises about 87% of total lipid. However, less expected is the general similarity of the fatty acid compositions of the phospholipids, especially PC
333
LIPIDS OF ARTEMIA CYSTS
TABLE 4 Identified fatty acids (I of total fatty acid) in the lipid classes and lipids of the Arfemia cysts from Great Salt Lake’ Fatty acid
14: 15:o 16:O 16: ln-9 16: In-7 16:2 17:o 16:3 18:O 18: In-9 18: ln-7 18:2n-6 18:3n-3 18:4n-3 20: In-9 20: 3n-3 20: 4n-3 20: 5n-3 Identified peaks n-3 HUFA n-6 HUFA n-3 PUFA n-6 PUFA n-3/n-6 Saturated Monoenes PUFA ‘For abbreviations
Lipid class (Ohof total fatty acids) PC
PS
PI
PE
TNL
TL
0.7 0.4 16.7 n.s 3.8 1.1 nd 1.7 8.4 22.9 6.2 6.2 15.4 4.4 0.6 1.0 0.7 0.5 90.7 2.2 0 22.0 6.2 3.5 26.2 33.5 31.0
1.7 2.1 17.2 1.6 2.6 1.4 nd 1.6 8.6 19.8 5.6 3.3 5.2 0.6 1.3 nd nd 0.6 73.2 0.6 0 6.4 3.3 1.9 29.6 30.9 12.7
2.2 2.2 15.8 2.4 2.9 1.5 nd 1.7 9.9 19.4 6.4 3.8 7.0 nd 1.6 nd nd nd 76.8 0 0 7.0 3.8 1.8 30.1 32.7 14.0
1.1 0.9 12.3 ns 3.2 0.8 1.0 1.7 6.3 26.1 7.4 5.1 13.8 3.4 nd nd 0.6 nd 84.3 0.6 0 17.8 5.7 3.1 21.6 36.7 26.0
1.5 0.5 19.1 2.6 3.3 1.0 1.2 2.1 6.2 23.2 7.2 5.3 10.8 1.3 0.7 nd 0.4 0.4 86.8 0.8 0 12.9 5.3 2.4 28.5 37.0 21.3
1.4 2.5 20.6 0.1 4.1 0.8 0.1 1.2 6.7 22.8 1.5 5.4 11.9 2.1 0.4 0.5 nd nd 89.3 0.5 0 14.5 5.4 2.7 31.3 36.1 21.9
see Table 3.
and PE, and the total neutral lipids (TNL). In particular, the percentages of the PUFA 20: 52-3, 20: 4n-6, 18 : 3n-3 and 18 : 2n-6 were quite similar in PC and PE, and in TNL. The same situation holds for 18 : 1 and also for 16 : 0, but not for 16 : 1 where the percentage in phospholipids was less than in total neutral lipid. Thus the relatively low ratio of 16 : O/ 16 : 1 in the marine-type cysts was dictated more by the fatty acid composition of the neutral lipids than the phospholipids. The lack of differentiation in fatty acids of neutral versus polar lipids in Artenzia nauplii has also been reported recently by Webster and Love11 ( 199 1) . The situation described for marine-type fatty acid compositions holds also for the freshwater-type cysts in Table 4, with the exception that the freshwater-type cysts have notably higher percentages of 18 : 3n-3 and only traces of
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C20 PUFA in both neutral lipids and individual phospholipids. Nonetheless, the composition of the phospholipids continues to mirror that of the neutral lipids in freshwater-type cysts. This holds even for the exception in marinetype cysts, namely 16 : 1, which was no more abundant in the neutral lipid of freshwater-type cysts than it was in their phospholipids. Freshwater-type cysts, therefore, have a lower percentage of 16: 1 in their lipid than marine-type cysts and this, together with the somewhat higher percentages of 16 : 0 in lipids from freshwater-type as compared to marine-type cysts, generates a notable higher ratio of 16 : O/ 16 : 1 in the freshwater-type cysts. This supports previous conclusions that the ratio of 16: O/ 16: 1 is higher in lipid from freshwater-type than marine-type Artemia (Navarro et al., 1992). It may be concluded, therefore, that marine-type cysts differ from their freshwater-type counterparts in having increased levels of 16 : 1 and C20 PUFA and decreased levels of 18 : 3n-3. Furthermore, atthough neutral lipids in marine-type cysts are relatively elevated in 16 : 1 as compared to phospholipids, the fatty acid compositions of the TNL are similar to those of the major individual phospholipids in both types of cysts. Therefore, if as is generally accepted, the fatty acid composition of their TAG is readily altered by diet, then so is the composition of their phospholipids. This, together with the relatively low percentages of PUFA in the organisms’ phospholipids, contrasts with the general situation in aquatic animals, especially marine animals, whose phospholipids have relatively constant fatty acid compositions with percentages of PUFA frequently exceeding 30% of the total fatty acids (see e.g. Vaskovski, 1989). The variability and generally atypical nature of the fatty acid compositions of even the major membrane structural lipids, PC and PE, of Artemia further illustrate the problem of phenotypic plasticity of the genus as well as its unreliability as a satisfactory live food for culturing marine fish and crustacean larvae. ACKNOWLEDGEMENTS
J.C.N was supported by a post-doctoral grant from the “Plan para el perfeccionamiento de doctores y tecnologos” from the Spanish Ministry of Education and Science.
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,4quaculture, 109 ( 1993) 327-336 Elsevier Science Publishers B.V., Amsterdam AQUA 50037
The lipids of the cysts of freshwater- and marinetype Artemia J.C. Navarrob, F. Amatb and J.R. SargenV “N.E.R. C. Unit ofAquatic Biochemistry, School of Natural Sciences, Stirling University, Stirling, UK blnstituto de Acuicultura de Torre de la Sal (C.S.I.C.), Ribera de Cabanes (Castekin), Spain (Accepted 6 May 1992)
ABSTRACT
Navarro, J.C., Amat, F. and Sargent, J.R., 1993. The lipids of the cysts of freshwater- and marine-type Artemia. Aquaculture, 109: 321-336. Cysts of marine-type Artemia had approximately twice the level of total lipid per unit dry weight as cysts of freshwater-type Artemiu. Total lipid of freshwater-type cysts had higher percentages of pigments, cholesterol and free fatty acids and lower percentages of triacylglycerols than total lipid of marine-type cysts. The percentage of phospholipids in total lipids was the same for both types of cysts. The phospholipids and triacylglycerols of marine-type cysts had substantial levels of 20: G-3 and 20: 4n-6 whereas freshwater-type cysts lacked these fatty acids but had elevated levels of I8 : 3n-3. The ratio 16: O/ 16 : 1 was higher in lipids, especially neutral lipids, of marine-type than of freshwater-type cysts. Lipids of both types of cysts had relatively low percentages of polyunsaturated fatty acids, with the fatty acid composition of the major individual phospholipids and neutral lipids being broadly similar. Artemia nauplii are the most widely used larval food in aquaculture, so the significance of the results for the culture of marine fish and crustacean larvae is discussed in relation to the importance of considering absolute rather than relative levels of polyunsaturated fatty acids in Artemiu and the phenotypic plasticity of the genus.
INTRODUCTION
Newly hatched nauplii of the brine shrimp, Artemia sp., are the most widely used larval food in aquaculture but their nutritional value is not completely satisfactory for marine fish and prawn larvae. All stocks of Artemia contain 18: 3~3, some but not all contain 20: 5~2-3,and none contains 22: 6n-3 in more than trace amounts. Watanabe et al. ( 1978a, 1980) classified Artemia into two general groups, freshwater-type hernia devoid of 20: 5n-3 and thus only suitable for feeding to freshwater animals, and marine-type Artemia containing 20: $2-3 and allegedly suitable for feeding to marine animals. This Correspondence to: Dr. J.C. Navarro, Instituto 12595 Ribera de Cabanes, Castellh, Spain.
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classification was based on the assumption that freshwater animals can convert 18 : 3n-3 to 20: 5n-3 and thence to 22 : 6n-3, and that marine animals cannot convert 18 : 3n-3 to 20: k-3 but can convert 20: h-3 to 22 : 6n-3. Since then, many studies have been devoted to characterising Artemiustocks from different geographical origins from the point of view of the fatty acid compositions of their lipid. High variability among strains and also among batches of the same strain has been reported. Nonetheless, when 18 : 3n-3 is present in high amounts, 20: h-3 is generally absent or present in very low amounts (Leger et al., 1986) in line with the freshwater-marine distinction described above. Consequently the distinction continues to be useful as a general guide, although for quantitative nutritional studies causes of variations in the quality of Artemia become important. Navarro et al. ( 1992 ) found no clear correlation between the polyunsaturated fatty acid (PUFA) composition of Artemia cysts and the coastal/inland location of their saline habitats. However, Lavens et al. ( 1989) and Navarro and Amat ( 1992) demonstrated that the diet of broodstock Artemia influenced the fatty acid composition of both cysts and newly hatched nauplii. Navarro et al. ( 1991) showed that approximately 80% of the total lipid of Artemia cysts was neutral lipid, which is strongly influenced by diet in most animals (Love, 1980; Gurr and Hat-wood, 199 1). Watanabe et al. ( 1978b), Sakamoto et al. ( 1982) and Leibowitz et al. ( 1987) have all reported fatty acid compositions of total neutral and total polar lipid fractions of Artemia nauplii. However, to our knowledge, fatty acid compositions of the individual phospholipids of Artemia cysts have not been described. We report here analysis of the levels and fatty acid compositions of neutral lipids and individual phospholipids in cysts from freshwater-type and marine-type Artemia and discuss the implications of the findings for aquaculture. MATERIALS AND METHODS
Cysts from the Artemia population of the “La Mata” coastal lagoon (Torrevieja, Alicante, Spain) harvested by the authors in July 1985 and belonging to a parthenogenetic diploid strain were selected as marine-type cysts (LMT ) . Cysts of commercial origin (A.T.P., S.A. ) known to be deficient in essential fatty acids for marine organisms were chosen as freshwater-type cysts (GSL) . The latter cysts were further analysed by electrophoretic techniques (Seidel et al., 1980; Requintina and Simpson, 1987) and were assessed as belonging to a population from Great Salt Lake (Utah, U.S.A.) (Navarro, 1990). Prior to lipid extraction, cyst samples were hydrated in seawater (salinity 34 g/l) until the cysts were observed to be spherical under a dissecting microscope. They were then decapsulated according to the procedures described in Bruggeman et al. ( 1979). After a thorough wash in distilled water, the cysts were blotted over soft paper tissue and divided into six aliquots, three of which
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were dried for 24 h at 110’ C to calculate the moisture content, the remaining three being used for lipid extraction. Preliminary studies showed no differences between the lipid classes of decapsulated and whole cysts. Therefore, decapsulated cysts were analysed here because their lipids were easier to extract, Lipids were extracted using the method of Folch et al. ( 1957), quantified gravimetrically using an analytical balance (Mettler H 64) and stored at 20’ C at a concentration of 10 mg/ml in chloroform:methanol (2 : 1, V.-V)containing 0.0 1% ( WY) of butylated hydroxytoluene (BHT) as antioxidant. Before sealing the vials, nitrogen was flushed through the samples to create an anoxic atmosphere. Extractions were carried out in triplicate. Lipid classes were analysed quantitatively using double development, high performance thin layer chromatography (HPTLC ) on 10 x 10 cm plates of silica gel 60 (E. Merck, Darmstadt, Germany) as detailed by Olsen and Henderson ( 1989). Individual samples were analysed in quintuplicate with appropriate lipid standards and blanks. After development, the plates were charred with a copper acetate-phosphoric acid reagent (Fewster et al., 1969 ) and analysed by quantitative scanning densitometry (Olsen and Henderson, 1989). Individual lipid classes were separated on 20 x 20 cm plates of silica gel 60 (E. Merck, Darmstadt, Germany) using the polar solvent system of Olsen and Henderson ( 1989), detected by spraying with 0.1% 2’ ,7’-dichlorofluorescein and subjected to acid-catalysed transmethylation (Christie, 1982 ) after adding 19 : 0 as an internal standard. Fatty acid methyl esters were extracted from the transmethylation reaction with hexane:diethyl ether ( 1: 1, v:v) and purified on 20 x 20 cm silica gel G 60 plates using hexane:diethyl ether:acetic acid (85 : 15 : 1.5, VXV) as solvent. Fatty acid methyl esters were finally analysed by high resolution capillary gas-liquid chromatography as detailed previously (Navarro et al., 1992). Total lipids and lipid classes were analysed using a Student t test (Zar, 1984), at a 95% (PC 0.05) confidence level. Prior to statistical analyses, percentage compositions were arcsine transformed (Zar, 1984). RESULTS
AND DISCUSSION
Table 1 shows that cysts from the marine-type Artemia (LMT) had twice the total lipid per unit dry weight as the freshwater-type samples (GSL). Leger et al. ( 1986) reported that mean total lipid levels of Artemia nauplii accounted for 18.92 + 4.5% of the dry weight. Even though deviations can be expected, freshwater-type Artemia samples tend to have lower total lipid levels than the marine-type samples (Navarro et al., 1992). Moreover, lipid levels in cysts are known to be lower than in nauplii (Dutrieu, 1960; Schauer et al., 1980; Navarro et al., 1991). Nonetheless, the value of 9.2% found here
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TABLE 1 General lipid content of the cysts of freshwater (GSL)- and marine (LMT)-type Type of lipid
GSL
LMT
Total lipids (% dry weight) Neutral lipids/polar lipids n-3 HUFA (mg/g dry weight ) Fatty acids (mg/g dry weight)
9.2 f 0.2a 6.5 0.3 54.8 + 4.0a
17.9+0.4b 6.4 10.2 157.6f2.0b
Artemia cysts’.’
‘Data are means k standard deviations of triplicates, except for the ratio neutral/polar and n-3 HUFA which were calculated directly from means in Tables 2, 3 and 4. *Within the same row, different letters denote significant differences (PcO.05). When no letters are present statistical analyses were not performed. TABLE 2 Lipid classes in freshwater (GSL)- and marine (LMT)-type Lipid class
I of total lipids GSL
Polar Sphingomyelin/ lysophosphatidylcholine Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid/ cardiolipin Phosphatidylethanolamine Neutral Pigments Cholesterol Free fatty acids Triacylglycerols Cholesterol esters Polar lipids Neutral lipids
decapsulated Artemia cysts’ mg/g dry weight
LMT
GSL
LMT
0.5 i 0.2a
0.4+0.la
0.7 f O.Oa
0.7kO.la
7.3kO.la 0.2kO.la 1.6kO.Oa
7.5+0.6a 0.3kO.Oa 1.5kO.la
7.2fO.Oa 0.2kO.la 1.5If:O.Oa
13.52 I.lb 0.5kO.Ob 2.8+0.2b
0.5kO.la 2.220.3a
0.7+O.Ob 3.2kO.lb
0.6 + O.Oa 2.2+0.3a
1.3+0.lb 5.8+0.lb
32.4f l.Oa 12.5f0.2a 9.6 f0.5a 23.4+ 0.2a 9.7+0.7a
12.2k0.7b 9.2 ? 0.4b 5.4+ 1.5b 51.9+2.3b 7.8f 1.3a
26.2+0.1a 12.420.2a 9.7+0.5a 22.9 + 0.2a 9.9 + 0.8a
19.3+ 1.3b 16.8k0.7b 10.7k2.8a 95.7f3.9b 15.1 f 2.7b
12.7 k 0.7a 87.6+0.6a
13.6f l.Oa 86.6* l.Oa
12.510.5a Sl.Ot0.7a
24.7 k 1.3b 157.5?2.7b
‘Means in the same row and unit with different letters are significantly different (a=0.05).
for the total lipid in the freshwater-type GSL cysts is lower than anticipated. The lipid level in the marine-type LMT sample coincides with the mean value reported in the literature (LCger et al., 1986 ). Table 1 shows, from calculations using the lipid class compositional data in Table 2, that the ratio of polar/neutral lipids of the marine-type LMT and the freshwater-type GSL remain unchanged despite the the former having greater amounts of both neutral and polar lipids (Table 2 ) . Likewise Table 1
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CYSTS
331
shows, from calculations using the fatty acid compositional data of total lipid in Tables 3 and 4, (a) a substantially higher level of total fatty acids per unit dry weight in marine-type (LMT) as compared to freshwater-type (GSL) cysts; and (b) higher levels of n-3 highly unsaturated fatty acids (HUFA), i.e. n-3 PUFA of chain lengths > C20, in the marine-type (LMT) as compared to the freshwater-type (GSL) cysts. Therefore, on the basis of the nutritional provision of fatty acids, in general, and HUFA, in particular, to marine fish and crustacean larvae, the freshwater-type cysts are much inferior to marine-type cysts. This stems from their having a lower percentage of glycerides in total lipid, a lower percentage of n-3 HUFA in glycerides, and also a lower content of total lipid per unit weight, as compared to marine-type cysts. This illustrates the importance in larval nutrition of expressing HUFA levels in live foods on an absolute basis, e.g. mg of HUFA per g of dry weight, rather than simply on the basis of percent fatty acid compositions. Table 2 shows that some 13% of the total lipid of both freshwater- and marine-type cysts was present as polar lipids. Phosphatidylcholine (PC) followed by phosphatidylethanolamine (PE) were the major polar lipids present and, although some differences occurred in percentage compositions of individual polar lipids between freshwater-type and marine-type cysts, e.g. PE had a higher percentage in marine-type LMT than in freshwater-type GSL, the differences were not marked. The situation for neutral lipids, which accounted for 87% of the total lipid in both GSL and LMT, is different. Thus, the marine-type LMT had twice the percentage of triacylglycerols (TAG) in total lipids as the freshwater-type GSL. In contrast, GSL had a markedly higher percentage of pigments and significantly higher percentages of both cholesterol and free fatty acids than LMT. That is, the total lipid of the freshwatertype GSL had a higher percentage of non-saponifiable lipid and conversely a lower percentage of fatty acids than that of the marine-type LMT. In terms of providing fatty acids, the lipid of GSL is clearly inferior to that of LMT. However, when the data in Table 2 are expressed as per g dry weight of cysts, so taking account of the different absolute lipid levels noted in Table 1, the amounts of pigments, cholesterol and free fatty acids are more similar in the two types of cysts (although significant differences do occur in the levels of pigments and cholesterol). At the same time, the levels of individual phospholipids, especially PC and PE, and TAG are markedly elevated in the marine-type LMT as compared to the freshwater-type GSL. The data for the fatty acid compositions of total lipid from marine-type (Table 3 ) and freshwater-type (Table 4 ) cysts conform to earlier analyses for Artemia total lipid (Watanabe et al., 1978a,b, 1983; LRger et al., 1986; Navarro, 1990). Thus, the freshwater-type (GSL, Table 4) had 18 : 3n-3, 18 : 2n6 and some 18 : 4n-3 as major PUFA but only traces of C20 PUFA. In contrast, the marine-type (LMT, Table 3) had 20: 5n-3 and 20: 4n-6 as well as 18 : 3n-3, I8 : 2n-6 and 18 : 4n-3 as major PUFA. Tables 3 and 4, however, ex-
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ET AL.
TABLE 3 Fatty acid composition Mata’
of the lipid classes and total lipids of the marine-type Arfemia cysts from La
Fatty acid
Lipid class (O/oof total fatty acids)
14:o 15:o 16:0 16: ln-9 16: In-7 16:2 16:3 18:O 18: In-9 18: In-7 18:2n-6 18:3n-3 18:4n-3 20:o 20: In-9 20:4n-6
20:4n-3 20: 5n-3 22:ln-I1 22: 5n-3 Identified peaks n-3 HUFA n-6 HUFA n-3 PUFA n-6 PUFA n-3/n-6 Saturated Monoenes PUFA
PC
PS
PI
PE
TNL
TL
0.6 0.8 13.3 1.7 6.8 1.4 1.7 5.8 28.4 8.4 3.9 1.7 I.1 nd nd 1.3 nd 5.4 nd nd 82.3 5.4 1.3 8.2 5.2 1.6 20.5 45.3 16.5
2.6 2.6 15.3 3.2 3.8 1.7 1.6 9.3 21.8 5.3 2.9 4.1 nd 0.5 1.1 nd nd 2.1 0.6 1.1 79.7 3.3 0 7.4 2.9 2.6 30.3 35.8 13.6
1.8 2.2 11.8 2.5 3.7 1.8 1.4 10.5 21.9 9.5 3.4 3.6 nd nd 1.4 2.1 nd 4.6 nd 1.0 83.2 5.6 2.1 9.2 5.5 1.7 26.3 39.0 16.9
0.9 1.5 8.9 ns 7.7 1.7 1.6 5.7 33.6 11.3 4.1 2.8 nd nd 0.9 2.3 nd 5.4 nd nd 88.4 5.4 2.3 8.2 6.4 1.3 17.0 53.5 17.9
1.7 0.8 15.2 1.8 12.2 1.4 2.4 3.0 26.8 11.6 4.5 2.6 1.1 nd 0.7 0.9 0.3 5.2 nd nd 92.2 5.5 0.9 9.2 5.4 1.7 20.7 53.1 18.4
1.3 0.6 13.8 ns 12.5 1.5 2.5 3.5 24.7 10.0 4.7 2.7 1.3 nd 0.5 1.2 tr 6.5 0.1 nd 87.4 6.5 1.2 10.5 5.9 1.8 19.2 47.8 20.4
Ins, not separated from the other monoene; nd, not detected; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; TNL, total neutral lipids; TL. total lipids.
tend previous analyses by documenting the fatty acid analyses of individual phospholipids. All of the phospholipids listed in Table 2 were analysed but, for the sake of clarity and because no obviously notable features were present, the data for the “unresolved pairs” sphingomyelin + lysophosphatidylcholine and phosphatidic acid + cardiolipin are omitted. The fatty acid compositions of total lipid and total neutral lipid were similar in the marine-type LMT (Table 3). This is to be expected since neutral lipid comprises about 87% of total lipid. However, less expected is the general similarity of the fatty acid compositions of the phospholipids, especially PC
333
LIPIDS OF ARTEMIA CYSTS
TABLE 4 Identified fatty acids (I of total fatty acid) in the lipid classes and lipids of the Arfemia cysts from Great Salt Lake’ Fatty acid
14: 15:o 16:O 16: ln-9 16: In-7 16:2 17:o 16:3 18:O 18: In-9 18: ln-7 18:2n-6 18:3n-3 18:4n-3 20: In-9 20: 3n-3 20: 4n-3 20: 5n-3 Identified peaks n-3 HUFA n-6 HUFA n-3 PUFA n-6 PUFA n-3/n-6 Saturated Monoenes PUFA ‘For abbreviations
Lipid class (Ohof total fatty acids) PC
PS
PI
PE
TNL
TL
0.7 0.4 16.7 n.s 3.8 1.1 nd 1.7 8.4 22.9 6.2 6.2 15.4 4.4 0.6 1.0 0.7 0.5 90.7 2.2 0 22.0 6.2 3.5 26.2 33.5 31.0
1.7 2.1 17.2 1.6 2.6 1.4 nd 1.6 8.6 19.8 5.6 3.3 5.2 0.6 1.3 nd nd 0.6 73.2 0.6 0 6.4 3.3 1.9 29.6 30.9 12.7
2.2 2.2 15.8 2.4 2.9 1.5 nd 1.7 9.9 19.4 6.4 3.8 7.0 nd 1.6 nd nd nd 76.8 0 0 7.0 3.8 1.8 30.1 32.7 14.0
1.1 0.9 12.3 ns 3.2 0.8 1.0 1.7 6.3 26.1 7.4 5.1 13.8 3.4 nd nd 0.6 nd 84.3 0.6 0 17.8 5.7 3.1 21.6 36.7 26.0
1.5 0.5 19.1 2.6 3.3 1.0 1.2 2.1 6.2 23.2 7.2 5.3 10.8 1.3 0.7 nd 0.4 0.4 86.8 0.8 0 12.9 5.3 2.4 28.5 37.0 21.3
1.4 2.5 20.6 0.1 4.1 0.8 0.1 1.2 6.7 22.8 1.5 5.4 11.9 2.1 0.4 0.5 nd nd 89.3 0.5 0 14.5 5.4 2.7 31.3 36.1 21.9
see Table 3.
and PE, and the total neutral lipids (TNL). In particular, the percentages of the PUFA 20: 52-3, 20: 4n-6, 18 : 3n-3 and 18 : 2n-6 were quite similar in PC and PE, and in TNL. The same situation holds for 18 : 1 and also for 16 : 0, but not for 16 : 1 where the percentage in phospholipids was less than in total neutral lipid. Thus the relatively low ratio of 16 : O/ 16 : 1 in the marine-type cysts was dictated more by the fatty acid composition of the neutral lipids than the phospholipids. The lack of differentiation in fatty acids of neutral versus polar lipids in Artenzia nauplii has also been reported recently by Webster and Love11 ( 199 1) . The situation described for marine-type fatty acid compositions holds also for the freshwater-type cysts in Table 4, with the exception that the freshwater-type cysts have notably higher percentages of 18 : 3n-3 and only traces of
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C20 PUFA in both neutral lipids and individual phospholipids. Nonetheless, the composition of the phospholipids continues to mirror that of the neutral lipids in freshwater-type cysts. This holds even for the exception in marinetype cysts, namely 16 : 1, which was no more abundant in the neutral lipid of freshwater-type cysts than it was in their phospholipids. Freshwater-type cysts, therefore, have a lower percentage of 16: 1 in their lipid than marine-type cysts and this, together with the somewhat higher percentages of 16 : 0 in lipids from freshwater-type as compared to marine-type cysts, generates a notable higher ratio of 16 : O/ 16 : 1 in the freshwater-type cysts. This supports previous conclusions that the ratio of 16: O/ 16: 1 is higher in lipid from freshwater-type than marine-type Artemia (Navarro et al., 1992). It may be concluded, therefore, that marine-type cysts differ from their freshwater-type counterparts in having increased levels of 16 : 1 and C20 PUFA and decreased levels of 18 : 3n-3. Furthermore, atthough neutral lipids in marine-type cysts are relatively elevated in 16 : 1 as compared to phospholipids, the fatty acid compositions of the TNL are similar to those of the major individual phospholipids in both types of cysts. Therefore, if as is generally accepted, the fatty acid composition of their TAG is readily altered by diet, then so is the composition of their phospholipids. This, together with the relatively low percentages of PUFA in the organisms’ phospholipids, contrasts with the general situation in aquatic animals, especially marine animals, whose phospholipids have relatively constant fatty acid compositions with percentages of PUFA frequently exceeding 30% of the total fatty acids (see e.g. Vaskovski, 1989). The variability and generally atypical nature of the fatty acid compositions of even the major membrane structural lipids, PC and PE, of Artemia further illustrate the problem of phenotypic plasticity of the genus as well as its unreliability as a satisfactory live food for culturing marine fish and crustacean larvae. ACKNOWLEDGEMENTS
J.C.N was supported by a post-doctoral grant from the “Plan para el perfeccionamiento de doctores y tecnologos” from the Spanish Ministry of Education and Science.
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