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Lipid and fatty acid composition of striped bass (Morone saxatilis) larvae during development
Comp. Biochern. Physiol. Vol. 111B, No. 4, pp. 665-674, 1995
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Pergamon
Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/95 $9.50 + 0.00
0305-0491(95)00011-9
Lipid and fatty acid composition of striped bass (Morone saxatilis) larvae during development Fu-Lin E. Chu and Sureyya Ozkizilcik School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062, U.S.A. Changes in lipid class, fatty acid composition, protein, and dry and wet weights of fertilized eggs and developing larvae of striped bass (Morone saxatilis) fed with the live food, Artemia, were investigated. A decrease of wet and dry weights and moisture was observed at the beginning of the larval stage. Larvae regained the original moisture level, and wet and dry weights increased steadily after feeding. Total lipids decreased from 190/lg/egg in fertilized eggs to 151 pg/egg during hatching and increased after feeding. When total lipid contents were expressed as a percentage of larval dry weight, a decline of lipid did not occur until after feeding. Total protein, on the other hand, increased right after feeding, hut there was some variation between days. Polar lipids increased significantly from 20/~g/egg at the egg stage to 199/lg/iarva at 26 days post-hatching (DPH), 2 days before the onset of metamorphosis, while neutral lipids declined from 175/~g/egg to 80 pg/larva during the same time period. Wax/steryl esters decreased from 150/1g/egg in fertilized eggs to 32/~g/larva at 26 DPH. Triacylglycerols dropped from 21/1g/egg to 15/ig/larva before feeding and increased gradually after feeding. In contrast, the level of cholesterol increased 2-3-fold. There was a significant increase of phospholipids, particularly phosphatidylcholine in larvae after feeding. The fatty acid composition of fish larvae was significantly influenced by the diet, Artemia. There was an indication of catabolism of endogenous eicosapentaenoic and docosahexaenoic acids during metamorphosis. Key words: Striped bass; Morone saxatilis; Egg; Larva; Metamorphosis; Larval development; Lipids; Fatty acids.
Comp. Biochem. Physiol. l l l B , 665-674, 1995.
Introduction From embryo (fertilized eggs) to adult, teleost fish go through a developmental sequence in which a larval fish metamorphoses to the adult form (juvenile). Metamorphosis in fish entails a series of complex behavioral, morphological, physiological and biochemical changes. Metamorphosis requires a substantial energy supply
to develop and form a definitive phenotype, and is costly (Balon, 1986). The duration of metamorphosis is species specific and may extend from a few days to several months depending on various environmental factors such as temperature and food availability (see Youson, 1988, for review). Exogenous feeding is particularly indispensable, in the case of planktotrophic larvae, because the endogenous nutrient supply is comparatively limited in embryos. Correspondence to: F.-L. E. Chu, Schoolof Marine Science, To understand the energetic sources and proVirginia Instituteof MarineScience,Collegeof William and Mary, Gloucester Point, VA23062, U.S.A. Tel. cesses, considerable studies have focused on the (804) 642-7349; Fax (804)642-7186. changes in biochemical composition that occur Received 10 September 1994; accepted 20 December 1994. during embryonic and larval development. Abbreviations: DW, dry weight; WE, wax esters; TAG, triacylglycerol; PC, phosphatidylcholine; PE, phos- Fraser et al. (1988) noted the utilization of phatidylethanolamine;PS, phosphatidylserine;PI, phos- phosphatidylcholine (PC) and triacylglycerol phatidylinositol;SPH, sphyngomyelin. (TAG) for embryogenesis in cod (Gadus 665
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F.-L. E. Chu and S. Ozkizilcik
morhua). Lipids and glycogen were the primary source for the catabolic demand in the rapidly developing egg of the red drum (Seiaenops ocellata), while protein did not contribute to catabolism (Vetter and Hodson, 1983). Both protein and lipid content declined in the southern hemisphere lamprey, Geotria australis, during metamorphosis (Bird and Potter, 1981). There was a significant decrease of lipid and carbohydrate contents in metamorphic larvae of Albula (Pfeiler and Luna, 1984). Depletion of body fat in coho salmon (Oncorhynchus kisutch) was found to be associated with smoltification (Woo et al., 1978). Similarly, a significant decline of total lipid content in the serum, liver, and light and dark muscle occurred during parr-smolt transformation in the steelhead trout, Salmo gairdneri (Sheridan et al., 1983). Results from most of these studies imply that there is an abrupt change in biochemistry and/or shift in metabolic reaction associated with embryonic and larval development. In most cases, lipids are one of the principal substrates for energy in either fertilized eggs or larvae during development. However, these studies examined changes that occurred only at a particular time period, not a complete sequence from embryogenesis to metamorphosis and early juvenile stage. In addition, most of these works concerned only the gross alteration of the total lipids, protein and carbohydrate. Only one study investigated the change of fatty acid during embryogenesis (Fraser et al., 1988). The polyunsaturated fatty acid (PUFA), eicosapentaenoic acid, is known to be essential for striped bass (Morone saxatilis) larval growth (Webster and Lovell, 1990). The lipid and fatty acid composition of the striped bass eggs was described by Eldridge et al. (1983). In order to better understand the energy metabolism in the larvae of striped bass, the present study investigated the changes in lipid classes and fatty acid composition in larvae of striped bass from fertilized eggs through the early juvenile stage.
Materials and Methods Fertilized eggs and larval fish Fertilized eggs (days 1-3) and post-hatching larvae (days 1-6) were obtained from King and Queen State Hatchery, Virginia and transferred to the laboratory in a container filled with culture water (3 psu). To follow the development through metamorphosis, about 6000 six DPH larvae were transferred to three conical tanks (capacity = 70 1, 2000 larvae/tank) in a recirculated system. Culture water (22°C, 3 psu) was recirculated through a 5 # m filter and
crushed oyster shells to eliminate uneaten food items, fecal particles and ammonia. Feeding was initiated on seven DPH using San Francisco Bay origin Artemia nauplii, and a food density of 5 nauplii/ml was maintained by periodically counting the number of nauplii in the culture water throughout the experiment. The Artemia cysts were hatched daily in 601 transparent tanks in 1/~m filtered estuarine water (24 ppt) at 28°C. Freshly hatched nauplii were collected on 150/~m filters, rinsed well and stored at 10°C for feeding. Before feeding was initiated, three replicates of pooled fertilized eggs or one to six DPH larvae (20 egg or larvae/sample) were collected for lipid and fatty acid assays. After the start of feeding, larvae were sampled from each of the three culture tanks (7-25 DPH, 20 larvae/ sample tank; 26-36 DPH, 5 larvae/sample tank), weighed and frozen the same day at - 8 0 ° C in chloroform and methanol (2:1, v/v) under nitrogen. Wet weights of fish eggs and larvae were measured before they were frozen for later lipid and fatty acid analyses. To determine moisture content [% moisture = 100 x (wet wt - dry wt/wet wt)] of fish egg and larvae, at each sampling, separate egg and larval samples (4-5 eggs or larvae/sample) were dried at 65°C for 24hr, after wet weights were obtained. Dry weights of fish egg and larva were calculated as [(dry wt (rag)=wet wt (mg) × (100% - % of moisture)].
Lipid and fatty acid analyses Total lipids were extracted from three replicates of pooled egg and larval samples (days i-3, 20 eggs/sample; 7-25 DPH, 20 larvae/ sample; 26-36 DPH, 5 larvae/sample) with chloroform-methanol-water (2:2:1) according to the method of Bligh and Dyer (1959). Total lipid content of the sample was measured by the method of Holland and Gabbott (1971). Neutral lipid classes were separated on S-III chromarods (Iatron Laboratories, Tokyo, Japan) using a solvent mixture of hexanediethyl ether-formic acid (85:15:0.04, v/v/v) and quantified with flame-ionization (TLCFID) using Iatroscan TH-10, MK-3 analyzer (Iatron Laboratories, Tokyo, Japan). Operating conditions were 2000 ml/min air flow, 0.73 kg/cm 3 hydrogen pressure and the scan speed was 3.1 mm/sec. Peak area determinations were performed by computer analysis (T DataScan, RSS Inc., Bemis, TN). Quantities of each lipid class were determined by comparison with standard curves constructed for each standard (1, 5, 10 and 20#g) and expressed as #g lipid/egg-larva or % of egg or larval dry weight. When the total lipid content of the samples was calculated by adding the quantities of individual lipid classes together, the results were agreeable
Lipids in striped bass larvae
with the measurements obtained from colorimetric method of Holland and Gabbott (1971). Separation of polar lipids was performed on HPTLC plates precoated with silica gel grade G (Whatman) due to the unsatisfactory separation of phospholipids on chromarods (Banerjee et al., 1985). A solvent mixture of methyl acetate-isopropanol--chloroform-methanol-0.25% KC1 (25:25:25:10:9, v/v/v/v/v) gave a clear separation of phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylinositol, phosphatidylserine, lyso-phosphatidylcholine and sphyngolipids (Vitiello and Zanetta, 1978). Plates were dried briefly and charred with 3% cupric acetate in 8% aqueous phosphoric acid at 180°C for 20min (Fewster et al., 1969). Lipid classes were quantified with a GS-300 Transmittance/reflectance scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). Known quantities of purified standards were run in parallel on the same plate and the response factor of each standard was calculated. The results were corrected with the response factors derived from purified standards and expressed as #g/egg-larva or % of dry weight. All the lipid standards were obtained from Sigma Chemical Co. (St. Louis, MO), except cetyl palmitate was provided by Dr A. R. Place of the Center of Marine Biotechnology, Baltimore, MD. To determine fatty acid composition, total lipids were transesterified with methanol and boron trifluoride (Cosper and Ackman, 1983). Separation of the fatty acid methyl esters (FAME) was carried out on a gas liquid chromatograph (GLC Varian 3300) equipped with a flame ionization detector, using a fused silica capillary column coated with SP-2330 (30 m x 0.25 mm i.d., Supelco, Bellefonte, PA). The column was temperature-programmed from 120 to 180°C at 12°C/min and from 180 to 220°C at 6°C/min; injector and detector temperatures were 220 and 240°C, respectively; the flow rates of compressed air and hydrogen were 300 and 30ml/min, respectively; helium was used as the carrier gas (1.5 ml/min). Identification of FAMEs was based on the comparison of the sample component retention time with those of authentic standards. Purified menhaden oil was also used as a secondary external standard for identification. Identifications were further confirmed with an Extrel ELQ400-2 GC/MS system using chemical ionization. FAME peaks were quantified by computer analysis (Chromatochart-PC, Interactive Microware, Inc.) connected to the GLC. The results were corrected with the response factors of external standards (18:0, 21:0, 20:5n-3, 22:6n-3) and expressed as a percentage of the total fatty acid methyl esters.
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Total protein, total lipid, lipid classes and select fatty acids were included in the statistical analysis. Percentages were arc sin transformed while log10 transformation was employed for biochemical composition data. One-way ANOVA with replicates was employed for statistical comparisons between egg and larvae, and between the different days post-hatching larvae, and followed by pairwise multiple comparison of means (Tukey) using the Systat® statistical package. Since no significant difference (P > 0.01) was found in fatty acid content in fertilized eggs between 1 and 3 DPH, prefeeding larvae between 1 and 7 DPH, and postfeeding larvae between 33 and 36 DPH, the data within each of these time periods were pooled.
Results Initiation of metamorphosis in striped bass larvae usually occurs between 28 and 30 days post-hatching when cultured at 21°C (Rogers et al., 1977). Morphologically, the formation of dorsal fins is an indication of the beginning of metamorphosis. The climax proceeds for 3 4 days (Mansueti, 1958; Parker and Specker, 1990). During this period, the gut becomes enclosed, the anal and dorsal fins become defined and the fin rays develop. In the present study, formation of dorsal fin rays was first noted at 28 DPH and completed at 31 DPH. Wet weight, dry weight and moisture content (%) of fish eggs, pre-feeding and post-feeding larval stages are shown in Fig. 1. There was a significant (P <0.05) decrease in wet (4.9 +0.2-1.1 __+0.0mg) and dry (0.43 ± 0 . 1 0.25 + 0.1 mg) weights and moisture content (91.1 ___0.4~77.0 _ 0.9%) from egg to one DPH, pre-feeding larvae. However, the moisture content increased back to 84% in larvae and remained above 80% throughout. There was a slight decrease at 28 DPH, the day dorsal fin formation was first noted and 31 DPH, the day metamorphosis was assumed to be terminated. Similarly, wet and dry weights gradually and steadily increased after feeding was commenced. Protein and lipids accounted for 63/~g/egg (16% dry wt) and 190#g/egg (47% dry wt), respectively, of the fertilized eggs (Fig. 2A and B). In pre-feeding larvae (1-7 DPH), total lipids decreased to approximately 150 #g/larva (62% dry wt), and protein to 43/zg/larva (18% dry wt), resulting in a concomitant drop in dry weight. Total protein content of the fertilized eggs dropped during hatching and increased steadily with the start of active feeding, although there was some variation between days. There was a marked increase (P < 0.05) of
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F.-L. E. Chu and S. Ozkizilcik
protein in fish larvae, from 43 #g/larva (18% dry wt) before feeding to 161 #g/larva (45% dry wt) 11 days after feeding (Fig. 2A and B). Total lipid content of the larvae increased to 401 #g/larva at 26 DPH, 2 days before the onset of metamorphosis. The lipid content declined slightly with the beginning of metamorphosis and was followed by an increase towards the end of metamorphosis on 30 and 31 DPH. Protein levels in larvae during metamorphosis showed a trend similar to that of total lipids and appeared to stabilize at approximately 55% of the dry weight after metamorphosis was completed. Neutral lipids (WE + TAG + cholesterol) decreased from 46 to 14% dry wt, as polar lipids increased from 9 to 14% dry wt, after the start of feeding (Fig. 3B). When the data were presented as /~g/larva, TAG and cholesterol showed a significant increase in feeding larvae (Fig. 3A). The decrease of neutral lipids was almost entirely due to the reduction of WE which comprised about 78% (150 #g/egg) and 72% (110 #g/larva) of the total lipids in fertilized eggs and pre-feeding larvae, respectively. Wax esters gradually decreased throughout the development and only trace amounts were detected at 26 DPH. Triacylglycerol content (21/~g/egg) dropped during hatching and early development of the pre-feeding larvae. After the beginning of feeding, TAG content gradually increased to 140/~g/larva at 32 DPH and re-
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mained at approximately 15% of the total lipids thereafter. The level of cholesterol was relatively constant in eggs (5 #g/egg) and pre-feeding larvae (5.5 #g/larva), and increased steadily after feeding to about 130/~g/larva. Total polar lipid content of the larvae increased dramatically with the beginning of feeding (Fig. 4A and B). Phosphatidylcholine was the dominant component of the polar lipids (10/~g/egg), followed by phosphatidylethanolamine. Phosphatidylcholine content of fertilized eggs significantly decreased (P < 0.05) before hatching. Generally, all polar lipid classes increased post-feeding, with PC being the most prominent. However, at the beginning of metamorphosis (28 DPH), there was a slight drop in PC and PE content of the larvae, followed by a significant increase 1 day before the completion of metamorphosis (30 DPH). This trend was similar to those observed with protein and total lipids. The composition of major fatty acid components in the total lipids of fish larvae and the diet, Artemia, is shown in Table 1. Mono- and polyunsaturated fatty acids were the major classes of component fatty acids in fish larvae. The fatty acid composition of the diet was dominated by oleic acid (18:1n-9), followed by linolenic acid (18:3n-3) and palmitic acid (16:0). Oleic and linolenic acids accounted for 28.2 and 22.7% of the total fatty acids of Artemia. Eicosapentaenoic acid (20:5n-3, EPA)
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Days Fig. 1. Wet weight (mg), dry weight (mg) and moisture content (%) of striped bass eggs and larvae during hatching and development. Values represent the mean of three replicates + SE.
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Days Fig. 2. Protein (PRT) and total lipid (TL), neutral lipids (NL) and polar lipids (PL) (A, pg/egg-larva; B, % of dry wt) of striped bass eggs and larvae during hatching and development. Feeding was initiated at 8 days post-hatching. Each value represents the mean of three replicates 4-SE. Arrows indicate the beginning and the end of metamorphosis. The inset demonstrates the values for hatching eggs and early larvae at a smaller scale.
was unusually low in this batch of Artemia, and docosahexaenoic acid (22:6n-3, D H A ) was not detected. A significant change in fatty
acid composition of larvae was noted after feeding. The percentages of palmitic (16: 0), linoleic (18 : 2n-6), linolenic, stearic (18 : 0) and
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arachidonic (20:4n-6) acids increased post-feeding, The former three components increased to a level similar to that in the Artemia, while the latter two acids augmented to a level 2-fold
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greater than the diet. The percentages of palmitoleic acid (16:1n-7), EPA and DHA declined post-feeding. The change in palmitoleic acid reflected the diet level of that component. Both
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Days Fig. 3. Triacylglycerol (TAG), cholesterol (CHOL) and wax esters (WE) content (A, #g/egg-larva; B, % of dry wt) of striped bass eggs and larvae during hatching and development. Each value represents the mean of three replicates + SE.
Lipids in striped bass larvae
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Days Fig. 4. Phospholipid composition (A,/~g/egg-larva; B, % of dry wt) of striped bass eggs and larvae during hatching and development. Feeding was initiated at 8 days post-hatching. Each value represents the mean of three replicates + SE. Arrows indicate the beginning and the end of metamorphosis. The inset illustrates the values for hatching eggs and early larvae at a smaller scale.
EPA and D H A fell after feeding from 7.8 and 6.3% in fertilized eggs to 2.2 and 0.1%, respectively, at 33-36 D P H . The percentage of oleic
acid dropped slightly 4 days after feeding. There was no significant change thereafter and its level was similar to that of the diet.
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Discussion The beginning o f the larval stage in striped bass is usually m a r k e d by a period o f decreased length a n d / o r suspended growth (Rogers et al., 1977). The c o n c o m i t a n t decrease in wet and dry weights and moisture content in newly hatched pre-feeding larvae c o m p a r e d with fertilized eggs m a y be an indication o f shrinkage o f fish length and g r o w t h suspension o f the larvae before external feeding, a l t h o u g h larval length was not measured in our study. D u r i n g this period, endogenous feeding is the main source o f nutrients; therefore, t e m p o r a r y suspension o f growth m a y be necessary to preserve energy. Loss o f weight and shrinkage in length (or suspension o f growth) was also f o u n d in larvae o f southern lamprey, Geotria australis before m e t a m o r p h o sis (Bird and Potter, 1981). Protein and lipid are the principal nutrient sources o f fish eggs. The quantity o f protein and lipid in fish eggs varies greatly a m o n g fish species. Depending on the species, protein comprises a b o u t 13-85% o f the egg dry weight, and lipids range from 3 to 55% o f the egg dry weight (Love, 1970; Vuorela et al. 1979; K a i t a r a n t a and A c k m a n , 1981; Eldridge et al., 1983). F o r example, in the n o r t h e r n pike (Esox lucius), the m a t u r e eggs consist o f 26.4% protein and 6.2% lipids ( M e d f o r d and M a c k a y , 1977). In Baltic herring (Clupea harengus) roe protein comprises 82.5%, while lipid constitutes 2.4%, o f the dry weight (Vuorela et al., 1979; K a i t a r a n t a and A c k m a n , 1981). In red d r u m (Sciaenops ocellata), 1 g o f wet eggs contained 21.2 mg lipids, 19.7mg protein and 0 . 3 m g glycogen (Vetter and H o d s o n , 1983). According to Eldridge et al.
(1983), striped bass eggs are rich in lipids. The oil globule contributed to 55% o f the egg dry weight, while the yolk (which contains mainly lipoprotein) accounted for only 38% o f the egg dry weight. Similarly, as demonstrated in the present study, striped bass fertilized eggs had a m u c h higher content o f lipid than protein (50 vs. 16%). The energy requirements o f fertilized fish eggs are usually met by the use o f protein or lipids (Blaxter, 1969; Love, 1970; Vuorela et al., 1979; Vetter and H o d s o n , 1983; Fraser et al., 1988). In striped bass, a previous study (Eldridge et al., 1982) indicated that complete utilization o f yolk energies occurred in post-hatching larvae before exogenous feeding, whereas the egg oil globule persisted for extended periods from the embryonic stage to post-hatching. Complete oil globule absorption did not occur until 20-29 D P H . However, in the present study, loss o f total lipids, W E in particular, and gain o f protein concurred in the hatching larvae, from 7 to 16 D P H . There was also a slight d r o p in protein, T A G , PC and PE between 28 and 29 D P H . These results infer that, before and during metamorphosis, the larvae rely primarily on lipids for energy (Fig. 2A and B). Utilization o f protein, on the other hand, occurred only during hatching and metamorphosis. Similar results were obtained in a study o f energy metabolism in the rapidly developing eggs of red d r u m (Sciaenops ocellata); lipids were the prim a r y source for energy d e m a n d during development, and protein did not contribute to catabolism (Vetter and H o d s o n , 1983). Polar lipids are structural lipids used for m e m b r a n e synthesis. T o w a r d the onset o f metamorphosis,
Table I. Fatty acid composition (% of total fatty acid methyl esters) of the total lipids of fertilized eggs and larvae during hatching and development D12 D16 D20 D26 D27 D28 D29 D30 D31 D32 D33-D36 Diet Fatty acid Egg D1-D7 (4) (8) (12) (18) (19) (20) (21) (22) (23) (24) (25 31) (Artemia) 14:0 1.9 2.0 0.9 0.5 0.5 0.1 tr ---0.2 0.1 0.2 0.8 16:0 9.2 9.0 13.6 14.3 17.9 17.4 18.1 18.0 18.6 17.5 16.9 17.3 16.7 14.9 16:1n-7 23.8 23.7 26.1 17.9 12.0 8.6 8.1 7.8 7.6 7.9 8.0 8.1 8.4 9.5 16:2 1.9 2.0 1.9 1.5 1.0 0.4 0.3 0.4 0.2 0.4 0.4 0.4 0.4 1.4 18:0 1.2 1.1 1.7 3.7 6.9 7.6 7.9 8.8 9.9 8.7 7.7 8.5 7.9 3.8 18:1n-9 37.5 36.8 27.9 36.7 32.6 29.4 31.3 31.3 30.6 30.8 31.1 31.6 30.9 28.2 18:2n-6 1.8 1.8 1.2 2.9 3.9 4.9 5.5 5.6 5.6 5.4 5.5 5.5 5.6 6.8 18:3n-3 1.1 1.1 1.7 6.5 11.1 19.2 19.5 18.6 18.2 19.0 20.1 19.7 19.7 22.7 18:4n-3 2.1 1.9 0.6 2.0 1.9 2.1 2.1 1.6 1.4 1.6 2.5 2.2 2.4 3.6 20:1 1.1 1.0 0.8 0.8 0.9 0.5 -0.6 -0.2 -0.3 0.2 20:4n-6 0.8 0.8 0.3 0.6 1.6 1.9 1.9 2.8 2.4 2.7 2.3 2.2 2.2 0.9 20:4n-3 1.1 0.9 0.3 0.2 0.6 0.9 0.4 0.4 0.1 0.6 0.6 0.6 0.7 0.4 20:5n-3 7.8 7.6 2.5 5.9 4.4 2.7 2.9 3.0 3.3 3.5 3.1 2.8 2.7 1.4 22:5n-3 0.8 0.8 2.5 tr 0.1 -tr . . . . . 22:6n-3 6.3 6.9 2.3 5.1 3.8 0.7 0.5 0~2 0.4 0.7 0.2 0.1 0.1 -1. DI D7: pre-feeding (after hatching). 2. Day 8: initiation of feeding. 3. D12 D36: post-feeding. 4. Values represent mean of three replicates (20 eggs or 5~20 larvae/replicate). Numbers in parentheses indicate days after feeding.
Lipids in striped bass larvae the increase of protein and steady gain of polar lipids along with the decrease of neutral lipids signalized growth. The high content (78% of total lipids) of wax/steryl esters found in fish embryos (fertilized eggs) and larvae at the early stage is consistent with previous findings (Eldridge et al., 1983; Vetter and Hodson, 1983). Wax esters comprised 90 and 29% of the total lipids in unfertilized striped bass eggs and in developing red drum eggs, respectively. Apparently, WE are the primary energy source for larval development and growth. Our results also suggested that larvae tend to retain WE in early planktonic life, since a dramatic decrease did not happen until 16 DPH. Although buoyancy as a principal function of lipid has at times been discounted (Smith, 1957; Russell, 1976), the retention of WE for this purpose may be necessary for the larvae of estuarine and fresh water species (Vetter and Hodson, 1983; Phleger and Grigor, 1990). Therefore, WE may play a dual role in striped bass: to keep eggs and early stage larvae neutrally buoyant in nearly freshwater and to serve as an energy reserve for metamorphogenesis. Augmentation of cholesterol and the decrease of WE happened at the same time in post-feeding striped bass larvae. Cholesterol levels increased more than 3-fold at 26 D P H larvae compared with pre-feeding larvae. As suggested by Dergaleva and Shatunovskiy (1977), the increase in cholesterol maybe the result of the breakdown of sterol esters in the larvae or the dietary accumulation from Artemia which contains about 3 #g cholesterol per mg wet weight (Ozkizilcik and Chu, 1994a). However, cholesterol did not appear to be used in the process of morphogenesis, since its level was relatively constant throughout metamorphosis. The increase of polar lipids (phospholipids, Fig. 4A) after feeding indicates not only growth, but extensive assimilation of these lipids. It has been suggested that, unlike neutral lipids, which are too large for direct absorption and must be broken down prior to absorption, polar lipids are amphiphatic molecules that would be readily emulsified in the digestive tract and are likely to be absorbed through pinocytosis (Olsen et al., 1991). Catabolism of phosphohpids, PC in particular, was also noted during metamorphosis. The PC level dropped markedly at 29 DPH. PC has been found to be the only lipid class catabolized in the cod, Gadus morhua, during embryogenesis (Fraser et al., 1988). Similar results were found in this study showing that there was a significant drop in polar lipid content of fertilized striped bass eggs during hatching. The fatty acid composition o f the fish larvae was modified significantly by the diet, Artemia.
673
Substantial changes took place in certain fatty acid components (e.g. 16:0, 16:1n-7, 18:1n-9, 18:2n-6, 18:n-3, 20:1) after feeding was initiated. The percentages of these fatty acids either increased or decreased to a level similar to that of the diets. The selective usage of certain fatty acids for energy may also be the cause of the declines in some of the fatty acid components. This is most evident in EPA and DHA; significant decreases of these two components occurred at 26 DPH, 2 days before the beginning of metamorphosis. There was also an indication of active assimilation of EPA, stearic and arachidonic acids in fish larvae. The level of these three fatty acids was almost constant after 26 DPH, and 2-fold higher than the diet. No significant changes occurred in any of the fatty acids during metamorphosis (26-31 DPH). It has been well documented that the fatty acid composition of the live food, Artemia, varies among batches and sources. The EPA level in the Artemia used for the present study was low compared with our previous analyses (Ozkizilcik and Chu, 1994b). San Francisco Bay origin Artemia sp. usually contains 8-9% EPA of total fatty acid methyl esters (Webster and Lovell, 1990). In summary, the neutral lipids, wax esters and triacylglycerols were the primary energy source for energy during larval development and growth of striped bass. The larvae extensively assimilated phospholipids before the onset of metamorphosis. The fatty acid composition of the larvae was significantly affected by the diet. Some of the fatty acids, such as, EPA and DHA, which were abundant in eggs, were utilized during larval development. Acknowledgements--This work is a result of research
sponsored by the NOAA Officeof Sea Grant, U. S. Department of Commerce, under Grant No. NA90AAoD-SG045 to Virginia Graduate Marine Science Consortium. The authors wish to thank Drs R. Hale and K. Webb for their critical review of the manuscript and special thanks to R. Mothershead for the GC/MS analysis of fatty acids. Contribution no. 1907from the Virginia Institute of Marine Science, College of William and Mary.
References Balon E. K. (1986)Typesof feedingin the ontogenyof fishes and the life-historymodel.Environ. Biol. Fishes. 16, 11-24. Banerjee A. K., Ratnayake W. M. N. and Ackman R. G. (1985) Effect of oxalic acid impregnation of chromarods on the separation of phospholipids for determination by the latroscan TLC/FID. Lipids 20, 121 125. Bird D. J. and Potter I. C. (1981) Proximate body composition of the larval, metamorphosing and downstream migrant stages in the life cycle of the Southern Hemisphere lamprey, Geotria australis. Environ. BioL Fishes. 6, 285-297. Blaxter J. H. S. (1969) Development: eggs and larvae. In Fish Physiology (Edited by Hoar W. S. and Randall D. J.), pp. 177-252. Academic Press, New York.
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Bligh E. G. and Dyer W. M. (1959) A rapid method of lipid extraction and purification. Can. J. Biochem. Physiol. 35, 911-917. Cosper C. I. and Ackman R. G. (1983) Occurrence of cis-9,10-methylene-hexadecanoic acids in the lipids of immature and mature Fundulus heteroclitus (L.) and in roe. Comp. Biochem. Physiol. 75B, 6494554. Dergaleva Z. T. and Shatunovskiy M. I. (1977) Data on the lipid metabolism of the larvae and young of the striped bass, Morone saxatilis. J. Ichthyol. 17, 802 804. Eldridge M. B., Joseph J, D., Taberski K. M. and Seaborn G. T. (1983) Lipid and fatty acid composition of the endogenous energy sources of striped bass (Morone saxatilis) eggs. Lipids, 18, 510-513. Eldridge M. B., Whipple J. A. and Bowers M. J. (1982) Bioenergetics and growth of striped bass, Morone saxatilis, embryos and larvae. Fish. Bull. 80, 461~474. Fewster M. E., Burns B. J. and Mead J. F. (1969) Quantitative densitometric thin-layer chromatography of lipids using copper acetate reagent. J. Chromatogr. 43, 120-126. Fraser A. J., Gamble J. C. and Sargent J. R. (1988) Changes in lipid content, lipid class and fatty acid composition of developing eggs and unfed larvae of cod (Gadus morhua). Mar. Biol. 99, 307-313. Holland D. L, and Gabbott P. A. (1971) A micro-analytical scheme for the determination of protein, carbohydrate, lipid and RNA levels in marine invertebrate larvae. J. Mar. Biol. Assoc. U.K. 52, 6594568. Kaitaranta, J. K. and Ackman R. G. (1981) Total lipids and lipid classes of fish roe. Comp. Biochem. Physiol. 69B, 725-729. Love R. M. (1980) The Chemical Biology of Fishes, Vol. H, Advances 1968 1977. Academic Press, London. Mansueti R. (1958) Eggs, larvae and young of the striped bass, Morone saxatilis. Contribution number 112, Maryland Department of Research and Education. Medford B. A. and Mackay W. C. (1978) Protein and lipid content of gonads, liver and muscle of northern pike (Esox lucius) in relation to gonad growth. J. Fish. Res. Bd Can. 35, 213-219. Olsen R. E., Henderson R. J. and Pedersen T. (199l) The influence of dietary lipid classes on the fatty acid composition of small cod Gadus morhua L. juveniles reared in an enclosure in northern Norway. J. exp. Mar. Biol. EcoL 148, 59 76. Ozkizilcik S. and Chu F.-L. E. (1994a) Uptake and metabolism of liposomes by Artemia nauplii. Aquaculture 128, 131-141. Ozkizilcik S. and Chu F.-L. E. (1994b) Evaluation of omega-3 fatty acid enrichment of Artemia nauplii as
food for striped bass Morone saxatilis Walbaum larvae. J. Wrld Aquac. Soc. 25, 147 154. Parker S. J. and Specker J. L. (1990) Salinity and temperature on whole-animal thyroid hormone levels in larval and juvenile striped bass, Morone saxatilis. Fish Physiol. Biochem. 8, 507-514. Pfeiler E. and Luna A. (1984) Changes in biochemical composition and energy utilization during metamorphosis and leptocephalus larvae of the bonefish (Albula). Environ. Biol. Fishes. 10, 243-251. Phleger C. F. and Grigor (1990) Role of wax esters in determining buoyancy in Haplostethus atlanticus (Berysciformes: Trachichthyidae). Mar. Biol. 105, 229 233. Rogers B. A., Westin D. T. and Saila S. B. (1977) Life stage duration studies on Houston River striped bass Morone saxatilis (Walbaum). University of Rhode Island. Marine Technical Report Number 31. Russell F. S. (1976) The Eggs and Planktonic Stages of British Marine Fishes. Academic Press, New York. Sheridan M. A., Allen W. V. and Kerstetter T. H. (1983) Seasonal variations in the lipid composition of the steelhead trout, Salmo gairdneri Richardson, associated with the parr-smolt transformation. J. Fish Biol. 23, 125 134. Sheridan M. A., Allen W. V. and Kerstetter T. H. (1985) Changes in the fatty acid composition of steelhead trout, Salmo gairdneri Richardson, associated with parr-smolt transformation. Comp. Biochem. Physiol. 80B, 671 676. Smith S. (1957) Early development and hatching. In The Physiology of Fishes (Edited by Brown M. E.), Vol. 1, pp. 232-359. Academic Press, New York. Vetter R. D. and Hodson R. E. (1983) Energy metabolism in a rapidly developing imarine fish egg, the red drum (Sciaenops ocellata). Can. J. Fish. Aquat. Sci. 40, 6274534. Vitiello F. and Zanetta J.-P. (1978) Thin layer chromatography of phospholipids. J. Chromatogr. 166, 637-640. Vuorela R., Kaitaranta J. and Linko R. R. (1979) Proximate composition offish roe in relation to maturity. Can. Inst. Food Sci. Technol. J. 12, 186 188. Webster C. D. and Lovell R. T. (1990) Response of striped bass larvae fed brine shrimp from different sources containing different fatty acid composition. Aquaculture 90, 49451. Woo N., Bern H. A, and Nishioka R. S. (1978) Changes in body composition associated with smoltification and premature transfer to seawater in coho salmon (Onchorhynchus kisutch) and king salmon (O. tschawytscha). J. Fish Biol. 13, 421~,28. Youson J. H. (1988) First metamorphosis. In Fish Physiology (Edited by Hoar W. S. and Randall D. J.), Vol. X1, Part B, pp. 135-184. Academic Press, New York.
~
Pergamon
Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/95 $9.50 + 0.00
0305-0491(95)00011-9
Lipid and fatty acid composition of striped bass (Morone saxatilis) larvae during development Fu-Lin E. Chu and Sureyya Ozkizilcik School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062, U.S.A. Changes in lipid class, fatty acid composition, protein, and dry and wet weights of fertilized eggs and developing larvae of striped bass (Morone saxatilis) fed with the live food, Artemia, were investigated. A decrease of wet and dry weights and moisture was observed at the beginning of the larval stage. Larvae regained the original moisture level, and wet and dry weights increased steadily after feeding. Total lipids decreased from 190/lg/egg in fertilized eggs to 151 pg/egg during hatching and increased after feeding. When total lipid contents were expressed as a percentage of larval dry weight, a decline of lipid did not occur until after feeding. Total protein, on the other hand, increased right after feeding, hut there was some variation between days. Polar lipids increased significantly from 20/~g/egg at the egg stage to 199/lg/iarva at 26 days post-hatching (DPH), 2 days before the onset of metamorphosis, while neutral lipids declined from 175/~g/egg to 80 pg/larva during the same time period. Wax/steryl esters decreased from 150/1g/egg in fertilized eggs to 32/~g/larva at 26 DPH. Triacylglycerols dropped from 21/1g/egg to 15/ig/larva before feeding and increased gradually after feeding. In contrast, the level of cholesterol increased 2-3-fold. There was a significant increase of phospholipids, particularly phosphatidylcholine in larvae after feeding. The fatty acid composition of fish larvae was significantly influenced by the diet, Artemia. There was an indication of catabolism of endogenous eicosapentaenoic and docosahexaenoic acids during metamorphosis. Key words: Striped bass; Morone saxatilis; Egg; Larva; Metamorphosis; Larval development; Lipids; Fatty acids.
Comp. Biochem. Physiol. l l l B , 665-674, 1995.
Introduction From embryo (fertilized eggs) to adult, teleost fish go through a developmental sequence in which a larval fish metamorphoses to the adult form (juvenile). Metamorphosis in fish entails a series of complex behavioral, morphological, physiological and biochemical changes. Metamorphosis requires a substantial energy supply
to develop and form a definitive phenotype, and is costly (Balon, 1986). The duration of metamorphosis is species specific and may extend from a few days to several months depending on various environmental factors such as temperature and food availability (see Youson, 1988, for review). Exogenous feeding is particularly indispensable, in the case of planktotrophic larvae, because the endogenous nutrient supply is comparatively limited in embryos. Correspondence to: F.-L. E. Chu, Schoolof Marine Science, To understand the energetic sources and proVirginia Instituteof MarineScience,Collegeof William and Mary, Gloucester Point, VA23062, U.S.A. Tel. cesses, considerable studies have focused on the (804) 642-7349; Fax (804)642-7186. changes in biochemical composition that occur Received 10 September 1994; accepted 20 December 1994. during embryonic and larval development. Abbreviations: DW, dry weight; WE, wax esters; TAG, triacylglycerol; PC, phosphatidylcholine; PE, phos- Fraser et al. (1988) noted the utilization of phatidylethanolamine;PS, phosphatidylserine;PI, phos- phosphatidylcholine (PC) and triacylglycerol phatidylinositol;SPH, sphyngomyelin. (TAG) for embryogenesis in cod (Gadus 665
666
F.-L. E. Chu and S. Ozkizilcik
morhua). Lipids and glycogen were the primary source for the catabolic demand in the rapidly developing egg of the red drum (Seiaenops ocellata), while protein did not contribute to catabolism (Vetter and Hodson, 1983). Both protein and lipid content declined in the southern hemisphere lamprey, Geotria australis, during metamorphosis (Bird and Potter, 1981). There was a significant decrease of lipid and carbohydrate contents in metamorphic larvae of Albula (Pfeiler and Luna, 1984). Depletion of body fat in coho salmon (Oncorhynchus kisutch) was found to be associated with smoltification (Woo et al., 1978). Similarly, a significant decline of total lipid content in the serum, liver, and light and dark muscle occurred during parr-smolt transformation in the steelhead trout, Salmo gairdneri (Sheridan et al., 1983). Results from most of these studies imply that there is an abrupt change in biochemistry and/or shift in metabolic reaction associated with embryonic and larval development. In most cases, lipids are one of the principal substrates for energy in either fertilized eggs or larvae during development. However, these studies examined changes that occurred only at a particular time period, not a complete sequence from embryogenesis to metamorphosis and early juvenile stage. In addition, most of these works concerned only the gross alteration of the total lipids, protein and carbohydrate. Only one study investigated the change of fatty acid during embryogenesis (Fraser et al., 1988). The polyunsaturated fatty acid (PUFA), eicosapentaenoic acid, is known to be essential for striped bass (Morone saxatilis) larval growth (Webster and Lovell, 1990). The lipid and fatty acid composition of the striped bass eggs was described by Eldridge et al. (1983). In order to better understand the energy metabolism in the larvae of striped bass, the present study investigated the changes in lipid classes and fatty acid composition in larvae of striped bass from fertilized eggs through the early juvenile stage.
Materials and Methods Fertilized eggs and larval fish Fertilized eggs (days 1-3) and post-hatching larvae (days 1-6) were obtained from King and Queen State Hatchery, Virginia and transferred to the laboratory in a container filled with culture water (3 psu). To follow the development through metamorphosis, about 6000 six DPH larvae were transferred to three conical tanks (capacity = 70 1, 2000 larvae/tank) in a recirculated system. Culture water (22°C, 3 psu) was recirculated through a 5 # m filter and
crushed oyster shells to eliminate uneaten food items, fecal particles and ammonia. Feeding was initiated on seven DPH using San Francisco Bay origin Artemia nauplii, and a food density of 5 nauplii/ml was maintained by periodically counting the number of nauplii in the culture water throughout the experiment. The Artemia cysts were hatched daily in 601 transparent tanks in 1/~m filtered estuarine water (24 ppt) at 28°C. Freshly hatched nauplii were collected on 150/~m filters, rinsed well and stored at 10°C for feeding. Before feeding was initiated, three replicates of pooled fertilized eggs or one to six DPH larvae (20 egg or larvae/sample) were collected for lipid and fatty acid assays. After the start of feeding, larvae were sampled from each of the three culture tanks (7-25 DPH, 20 larvae/ sample tank; 26-36 DPH, 5 larvae/sample tank), weighed and frozen the same day at - 8 0 ° C in chloroform and methanol (2:1, v/v) under nitrogen. Wet weights of fish eggs and larvae were measured before they were frozen for later lipid and fatty acid analyses. To determine moisture content [% moisture = 100 x (wet wt - dry wt/wet wt)] of fish egg and larvae, at each sampling, separate egg and larval samples (4-5 eggs or larvae/sample) were dried at 65°C for 24hr, after wet weights were obtained. Dry weights of fish egg and larva were calculated as [(dry wt (rag)=wet wt (mg) × (100% - % of moisture)].
Lipid and fatty acid analyses Total lipids were extracted from three replicates of pooled egg and larval samples (days i-3, 20 eggs/sample; 7-25 DPH, 20 larvae/ sample; 26-36 DPH, 5 larvae/sample) with chloroform-methanol-water (2:2:1) according to the method of Bligh and Dyer (1959). Total lipid content of the sample was measured by the method of Holland and Gabbott (1971). Neutral lipid classes were separated on S-III chromarods (Iatron Laboratories, Tokyo, Japan) using a solvent mixture of hexanediethyl ether-formic acid (85:15:0.04, v/v/v) and quantified with flame-ionization (TLCFID) using Iatroscan TH-10, MK-3 analyzer (Iatron Laboratories, Tokyo, Japan). Operating conditions were 2000 ml/min air flow, 0.73 kg/cm 3 hydrogen pressure and the scan speed was 3.1 mm/sec. Peak area determinations were performed by computer analysis (T DataScan, RSS Inc., Bemis, TN). Quantities of each lipid class were determined by comparison with standard curves constructed for each standard (1, 5, 10 and 20#g) and expressed as #g lipid/egg-larva or % of egg or larval dry weight. When the total lipid content of the samples was calculated by adding the quantities of individual lipid classes together, the results were agreeable
Lipids in striped bass larvae
with the measurements obtained from colorimetric method of Holland and Gabbott (1971). Separation of polar lipids was performed on HPTLC plates precoated with silica gel grade G (Whatman) due to the unsatisfactory separation of phospholipids on chromarods (Banerjee et al., 1985). A solvent mixture of methyl acetate-isopropanol--chloroform-methanol-0.25% KC1 (25:25:25:10:9, v/v/v/v/v) gave a clear separation of phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylinositol, phosphatidylserine, lyso-phosphatidylcholine and sphyngolipids (Vitiello and Zanetta, 1978). Plates were dried briefly and charred with 3% cupric acetate in 8% aqueous phosphoric acid at 180°C for 20min (Fewster et al., 1969). Lipid classes were quantified with a GS-300 Transmittance/reflectance scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). Known quantities of purified standards were run in parallel on the same plate and the response factor of each standard was calculated. The results were corrected with the response factors derived from purified standards and expressed as #g/egg-larva or % of dry weight. All the lipid standards were obtained from Sigma Chemical Co. (St. Louis, MO), except cetyl palmitate was provided by Dr A. R. Place of the Center of Marine Biotechnology, Baltimore, MD. To determine fatty acid composition, total lipids were transesterified with methanol and boron trifluoride (Cosper and Ackman, 1983). Separation of the fatty acid methyl esters (FAME) was carried out on a gas liquid chromatograph (GLC Varian 3300) equipped with a flame ionization detector, using a fused silica capillary column coated with SP-2330 (30 m x 0.25 mm i.d., Supelco, Bellefonte, PA). The column was temperature-programmed from 120 to 180°C at 12°C/min and from 180 to 220°C at 6°C/min; injector and detector temperatures were 220 and 240°C, respectively; the flow rates of compressed air and hydrogen were 300 and 30ml/min, respectively; helium was used as the carrier gas (1.5 ml/min). Identification of FAMEs was based on the comparison of the sample component retention time with those of authentic standards. Purified menhaden oil was also used as a secondary external standard for identification. Identifications were further confirmed with an Extrel ELQ400-2 GC/MS system using chemical ionization. FAME peaks were quantified by computer analysis (Chromatochart-PC, Interactive Microware, Inc.) connected to the GLC. The results were corrected with the response factors of external standards (18:0, 21:0, 20:5n-3, 22:6n-3) and expressed as a percentage of the total fatty acid methyl esters.
667
Total protein, total lipid, lipid classes and select fatty acids were included in the statistical analysis. Percentages were arc sin transformed while log10 transformation was employed for biochemical composition data. One-way ANOVA with replicates was employed for statistical comparisons between egg and larvae, and between the different days post-hatching larvae, and followed by pairwise multiple comparison of means (Tukey) using the Systat® statistical package. Since no significant difference (P > 0.01) was found in fatty acid content in fertilized eggs between 1 and 3 DPH, prefeeding larvae between 1 and 7 DPH, and postfeeding larvae between 33 and 36 DPH, the data within each of these time periods were pooled.
Results Initiation of metamorphosis in striped bass larvae usually occurs between 28 and 30 days post-hatching when cultured at 21°C (Rogers et al., 1977). Morphologically, the formation of dorsal fins is an indication of the beginning of metamorphosis. The climax proceeds for 3 4 days (Mansueti, 1958; Parker and Specker, 1990). During this period, the gut becomes enclosed, the anal and dorsal fins become defined and the fin rays develop. In the present study, formation of dorsal fin rays was first noted at 28 DPH and completed at 31 DPH. Wet weight, dry weight and moisture content (%) of fish eggs, pre-feeding and post-feeding larval stages are shown in Fig. 1. There was a significant (P <0.05) decrease in wet (4.9 +0.2-1.1 __+0.0mg) and dry (0.43 ± 0 . 1 0.25 + 0.1 mg) weights and moisture content (91.1 ___0.4~77.0 _ 0.9%) from egg to one DPH, pre-feeding larvae. However, the moisture content increased back to 84% in larvae and remained above 80% throughout. There was a slight decrease at 28 DPH, the day dorsal fin formation was first noted and 31 DPH, the day metamorphosis was assumed to be terminated. Similarly, wet and dry weights gradually and steadily increased after feeding was commenced. Protein and lipids accounted for 63/~g/egg (16% dry wt) and 190#g/egg (47% dry wt), respectively, of the fertilized eggs (Fig. 2A and B). In pre-feeding larvae (1-7 DPH), total lipids decreased to approximately 150 #g/larva (62% dry wt), and protein to 43/zg/larva (18% dry wt), resulting in a concomitant drop in dry weight. Total protein content of the fertilized eggs dropped during hatching and increased steadily with the start of active feeding, although there was some variation between days. There was a marked increase (P < 0.05) of
668
F.-L. E. Chu and S. Ozkizilcik
protein in fish larvae, from 43 #g/larva (18% dry wt) before feeding to 161 #g/larva (45% dry wt) 11 days after feeding (Fig. 2A and B). Total lipid content of the larvae increased to 401 #g/larva at 26 DPH, 2 days before the onset of metamorphosis. The lipid content declined slightly with the beginning of metamorphosis and was followed by an increase towards the end of metamorphosis on 30 and 31 DPH. Protein levels in larvae during metamorphosis showed a trend similar to that of total lipids and appeared to stabilize at approximately 55% of the dry weight after metamorphosis was completed. Neutral lipids (WE + TAG + cholesterol) decreased from 46 to 14% dry wt, as polar lipids increased from 9 to 14% dry wt, after the start of feeding (Fig. 3B). When the data were presented as /~g/larva, TAG and cholesterol showed a significant increase in feeding larvae (Fig. 3A). The decrease of neutral lipids was almost entirely due to the reduction of WE which comprised about 78% (150 #g/egg) and 72% (110 #g/larva) of the total lipids in fertilized eggs and pre-feeding larvae, respectively. Wax esters gradually decreased throughout the development and only trace amounts were detected at 26 DPH. Triacylglycerol content (21/~g/egg) dropped during hatching and early development of the pre-feeding larvae. After the beginning of feeding, TAG content gradually increased to 140/~g/larva at 32 DPH and re-
75
I
mained at approximately 15% of the total lipids thereafter. The level of cholesterol was relatively constant in eggs (5 #g/egg) and pre-feeding larvae (5.5 #g/larva), and increased steadily after feeding to about 130/~g/larva. Total polar lipid content of the larvae increased dramatically with the beginning of feeding (Fig. 4A and B). Phosphatidylcholine was the dominant component of the polar lipids (10/~g/egg), followed by phosphatidylethanolamine. Phosphatidylcholine content of fertilized eggs significantly decreased (P < 0.05) before hatching. Generally, all polar lipid classes increased post-feeding, with PC being the most prominent. However, at the beginning of metamorphosis (28 DPH), there was a slight drop in PC and PE content of the larvae, followed by a significant increase 1 day before the completion of metamorphosis (30 DPH). This trend was similar to those observed with protein and total lipids. The composition of major fatty acid components in the total lipids of fish larvae and the diet, Artemia, is shown in Table 1. Mono- and polyunsaturated fatty acids were the major classes of component fatty acids in fish larvae. The fatty acid composition of the diet was dominated by oleic acid (18:1n-9), followed by linolenic acid (18:3n-3) and palmitic acid (16:0). Oleic and linolenic acids accounted for 28.2 and 22.7% of the total fatty acids of Artemia. Eicosapentaenoic acid (20:5n-3, EPA)
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was unusually low in this batch of Artemia, and docosahexaenoic acid (22:6n-3, D H A ) was not detected. A significant change in fatty
acid composition of larvae was noted after feeding. The percentages of palmitic (16: 0), linoleic (18 : 2n-6), linolenic, stearic (18 : 0) and
670
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arachidonic (20:4n-6) acids increased post-feeding, The former three components increased to a level similar to that in the Artemia, while the latter two acids augmented to a level 2-fold
250
greater than the diet. The percentages of palmitoleic acid (16:1n-7), EPA and DHA declined post-feeding. The change in palmitoleic acid reflected the diet level of that component. Both
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4 E
0
2 3 1 2 3 4 5 6 7
12
16
Egg
20
2627282980518283848586 Larva
Days Fig. 4. Phospholipid composition (A,/~g/egg-larva; B, % of dry wt) of striped bass eggs and larvae during hatching and development. Feeding was initiated at 8 days post-hatching. Each value represents the mean of three replicates + SE. Arrows indicate the beginning and the end of metamorphosis. The inset illustrates the values for hatching eggs and early larvae at a smaller scale.
EPA and D H A fell after feeding from 7.8 and 6.3% in fertilized eggs to 2.2 and 0.1%, respectively, at 33-36 D P H . The percentage of oleic
acid dropped slightly 4 days after feeding. There was no significant change thereafter and its level was similar to that of the diet.
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Discussion The beginning o f the larval stage in striped bass is usually m a r k e d by a period o f decreased length a n d / o r suspended growth (Rogers et al., 1977). The c o n c o m i t a n t decrease in wet and dry weights and moisture content in newly hatched pre-feeding larvae c o m p a r e d with fertilized eggs m a y be an indication o f shrinkage o f fish length and g r o w t h suspension o f the larvae before external feeding, a l t h o u g h larval length was not measured in our study. D u r i n g this period, endogenous feeding is the main source o f nutrients; therefore, t e m p o r a r y suspension o f growth m a y be necessary to preserve energy. Loss o f weight and shrinkage in length (or suspension o f growth) was also f o u n d in larvae o f southern lamprey, Geotria australis before m e t a m o r p h o sis (Bird and Potter, 1981). Protein and lipid are the principal nutrient sources o f fish eggs. The quantity o f protein and lipid in fish eggs varies greatly a m o n g fish species. Depending on the species, protein comprises a b o u t 13-85% o f the egg dry weight, and lipids range from 3 to 55% o f the egg dry weight (Love, 1970; Vuorela et al. 1979; K a i t a r a n t a and A c k m a n , 1981; Eldridge et al., 1983). F o r example, in the n o r t h e r n pike (Esox lucius), the m a t u r e eggs consist o f 26.4% protein and 6.2% lipids ( M e d f o r d and M a c k a y , 1977). In Baltic herring (Clupea harengus) roe protein comprises 82.5%, while lipid constitutes 2.4%, o f the dry weight (Vuorela et al., 1979; K a i t a r a n t a and A c k m a n , 1981). In red d r u m (Sciaenops ocellata), 1 g o f wet eggs contained 21.2 mg lipids, 19.7mg protein and 0 . 3 m g glycogen (Vetter and H o d s o n , 1983). According to Eldridge et al.
(1983), striped bass eggs are rich in lipids. The oil globule contributed to 55% o f the egg dry weight, while the yolk (which contains mainly lipoprotein) accounted for only 38% o f the egg dry weight. Similarly, as demonstrated in the present study, striped bass fertilized eggs had a m u c h higher content o f lipid than protein (50 vs. 16%). The energy requirements o f fertilized fish eggs are usually met by the use o f protein or lipids (Blaxter, 1969; Love, 1970; Vuorela et al., 1979; Vetter and H o d s o n , 1983; Fraser et al., 1988). In striped bass, a previous study (Eldridge et al., 1982) indicated that complete utilization o f yolk energies occurred in post-hatching larvae before exogenous feeding, whereas the egg oil globule persisted for extended periods from the embryonic stage to post-hatching. Complete oil globule absorption did not occur until 20-29 D P H . However, in the present study, loss o f total lipids, W E in particular, and gain o f protein concurred in the hatching larvae, from 7 to 16 D P H . There was also a slight d r o p in protein, T A G , PC and PE between 28 and 29 D P H . These results infer that, before and during metamorphosis, the larvae rely primarily on lipids for energy (Fig. 2A and B). Utilization o f protein, on the other hand, occurred only during hatching and metamorphosis. Similar results were obtained in a study o f energy metabolism in the rapidly developing eggs of red d r u m (Sciaenops ocellata); lipids were the prim a r y source for energy d e m a n d during development, and protein did not contribute to catabolism (Vetter and H o d s o n , 1983). Polar lipids are structural lipids used for m e m b r a n e synthesis. T o w a r d the onset o f metamorphosis,
Table I. Fatty acid composition (% of total fatty acid methyl esters) of the total lipids of fertilized eggs and larvae during hatching and development D12 D16 D20 D26 D27 D28 D29 D30 D31 D32 D33-D36 Diet Fatty acid Egg D1-D7 (4) (8) (12) (18) (19) (20) (21) (22) (23) (24) (25 31) (Artemia) 14:0 1.9 2.0 0.9 0.5 0.5 0.1 tr ---0.2 0.1 0.2 0.8 16:0 9.2 9.0 13.6 14.3 17.9 17.4 18.1 18.0 18.6 17.5 16.9 17.3 16.7 14.9 16:1n-7 23.8 23.7 26.1 17.9 12.0 8.6 8.1 7.8 7.6 7.9 8.0 8.1 8.4 9.5 16:2 1.9 2.0 1.9 1.5 1.0 0.4 0.3 0.4 0.2 0.4 0.4 0.4 0.4 1.4 18:0 1.2 1.1 1.7 3.7 6.9 7.6 7.9 8.8 9.9 8.7 7.7 8.5 7.9 3.8 18:1n-9 37.5 36.8 27.9 36.7 32.6 29.4 31.3 31.3 30.6 30.8 31.1 31.6 30.9 28.2 18:2n-6 1.8 1.8 1.2 2.9 3.9 4.9 5.5 5.6 5.6 5.4 5.5 5.5 5.6 6.8 18:3n-3 1.1 1.1 1.7 6.5 11.1 19.2 19.5 18.6 18.2 19.0 20.1 19.7 19.7 22.7 18:4n-3 2.1 1.9 0.6 2.0 1.9 2.1 2.1 1.6 1.4 1.6 2.5 2.2 2.4 3.6 20:1 1.1 1.0 0.8 0.8 0.9 0.5 -0.6 -0.2 -0.3 0.2 20:4n-6 0.8 0.8 0.3 0.6 1.6 1.9 1.9 2.8 2.4 2.7 2.3 2.2 2.2 0.9 20:4n-3 1.1 0.9 0.3 0.2 0.6 0.9 0.4 0.4 0.1 0.6 0.6 0.6 0.7 0.4 20:5n-3 7.8 7.6 2.5 5.9 4.4 2.7 2.9 3.0 3.3 3.5 3.1 2.8 2.7 1.4 22:5n-3 0.8 0.8 2.5 tr 0.1 -tr . . . . . 22:6n-3 6.3 6.9 2.3 5.1 3.8 0.7 0.5 0~2 0.4 0.7 0.2 0.1 0.1 -1. DI D7: pre-feeding (after hatching). 2. Day 8: initiation of feeding. 3. D12 D36: post-feeding. 4. Values represent mean of three replicates (20 eggs or 5~20 larvae/replicate). Numbers in parentheses indicate days after feeding.
Lipids in striped bass larvae the increase of protein and steady gain of polar lipids along with the decrease of neutral lipids signalized growth. The high content (78% of total lipids) of wax/steryl esters found in fish embryos (fertilized eggs) and larvae at the early stage is consistent with previous findings (Eldridge et al., 1983; Vetter and Hodson, 1983). Wax esters comprised 90 and 29% of the total lipids in unfertilized striped bass eggs and in developing red drum eggs, respectively. Apparently, WE are the primary energy source for larval development and growth. Our results also suggested that larvae tend to retain WE in early planktonic life, since a dramatic decrease did not happen until 16 DPH. Although buoyancy as a principal function of lipid has at times been discounted (Smith, 1957; Russell, 1976), the retention of WE for this purpose may be necessary for the larvae of estuarine and fresh water species (Vetter and Hodson, 1983; Phleger and Grigor, 1990). Therefore, WE may play a dual role in striped bass: to keep eggs and early stage larvae neutrally buoyant in nearly freshwater and to serve as an energy reserve for metamorphogenesis. Augmentation of cholesterol and the decrease of WE happened at the same time in post-feeding striped bass larvae. Cholesterol levels increased more than 3-fold at 26 D P H larvae compared with pre-feeding larvae. As suggested by Dergaleva and Shatunovskiy (1977), the increase in cholesterol maybe the result of the breakdown of sterol esters in the larvae or the dietary accumulation from Artemia which contains about 3 #g cholesterol per mg wet weight (Ozkizilcik and Chu, 1994a). However, cholesterol did not appear to be used in the process of morphogenesis, since its level was relatively constant throughout metamorphosis. The increase of polar lipids (phospholipids, Fig. 4A) after feeding indicates not only growth, but extensive assimilation of these lipids. It has been suggested that, unlike neutral lipids, which are too large for direct absorption and must be broken down prior to absorption, polar lipids are amphiphatic molecules that would be readily emulsified in the digestive tract and are likely to be absorbed through pinocytosis (Olsen et al., 1991). Catabolism of phosphohpids, PC in particular, was also noted during metamorphosis. The PC level dropped markedly at 29 DPH. PC has been found to be the only lipid class catabolized in the cod, Gadus morhua, during embryogenesis (Fraser et al., 1988). Similar results were found in this study showing that there was a significant drop in polar lipid content of fertilized striped bass eggs during hatching. The fatty acid composition o f the fish larvae was modified significantly by the diet, Artemia.
673
Substantial changes took place in certain fatty acid components (e.g. 16:0, 16:1n-7, 18:1n-9, 18:2n-6, 18:n-3, 20:1) after feeding was initiated. The percentages of these fatty acids either increased or decreased to a level similar to that of the diets. The selective usage of certain fatty acids for energy may also be the cause of the declines in some of the fatty acid components. This is most evident in EPA and DHA; significant decreases of these two components occurred at 26 DPH, 2 days before the beginning of metamorphosis. There was also an indication of active assimilation of EPA, stearic and arachidonic acids in fish larvae. The level of these three fatty acids was almost constant after 26 DPH, and 2-fold higher than the diet. No significant changes occurred in any of the fatty acids during metamorphosis (26-31 DPH). It has been well documented that the fatty acid composition of the live food, Artemia, varies among batches and sources. The EPA level in the Artemia used for the present study was low compared with our previous analyses (Ozkizilcik and Chu, 1994b). San Francisco Bay origin Artemia sp. usually contains 8-9% EPA of total fatty acid methyl esters (Webster and Lovell, 1990). In summary, the neutral lipids, wax esters and triacylglycerols were the primary energy source for energy during larval development and growth of striped bass. The larvae extensively assimilated phospholipids before the onset of metamorphosis. The fatty acid composition of the larvae was significantly affected by the diet. Some of the fatty acids, such as, EPA and DHA, which were abundant in eggs, were utilized during larval development. Acknowledgements--This work is a result of research
sponsored by the NOAA Officeof Sea Grant, U. S. Department of Commerce, under Grant No. NA90AAoD-SG045 to Virginia Graduate Marine Science Consortium. The authors wish to thank Drs R. Hale and K. Webb for their critical review of the manuscript and special thanks to R. Mothershead for the GC/MS analysis of fatty acids. Contribution no. 1907from the Virginia Institute of Marine Science, College of William and Mary.
References Balon E. K. (1986)Typesof feedingin the ontogenyof fishes and the life-historymodel.Environ. Biol. Fishes. 16, 11-24. Banerjee A. K., Ratnayake W. M. N. and Ackman R. G. (1985) Effect of oxalic acid impregnation of chromarods on the separation of phospholipids for determination by the latroscan TLC/FID. Lipids 20, 121 125. Bird D. J. and Potter I. C. (1981) Proximate body composition of the larval, metamorphosing and downstream migrant stages in the life cycle of the Southern Hemisphere lamprey, Geotria australis. Environ. BioL Fishes. 6, 285-297. Blaxter J. H. S. (1969) Development: eggs and larvae. In Fish Physiology (Edited by Hoar W. S. and Randall D. J.), pp. 177-252. Academic Press, New York.
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Bligh E. G. and Dyer W. M. (1959) A rapid method of lipid extraction and purification. Can. J. Biochem. Physiol. 35, 911-917. Cosper C. I. and Ackman R. G. (1983) Occurrence of cis-9,10-methylene-hexadecanoic acids in the lipids of immature and mature Fundulus heteroclitus (L.) and in roe. Comp. Biochem. Physiol. 75B, 6494554. Dergaleva Z. T. and Shatunovskiy M. I. (1977) Data on the lipid metabolism of the larvae and young of the striped bass, Morone saxatilis. J. Ichthyol. 17, 802 804. Eldridge M. B., Joseph J, D., Taberski K. M. and Seaborn G. T. (1983) Lipid and fatty acid composition of the endogenous energy sources of striped bass (Morone saxatilis) eggs. Lipids, 18, 510-513. Eldridge M. B., Whipple J. A. and Bowers M. J. (1982) Bioenergetics and growth of striped bass, Morone saxatilis, embryos and larvae. Fish. Bull. 80, 461~474. Fewster M. E., Burns B. J. and Mead J. F. (1969) Quantitative densitometric thin-layer chromatography of lipids using copper acetate reagent. J. Chromatogr. 43, 120-126. Fraser A. J., Gamble J. C. and Sargent J. R. (1988) Changes in lipid content, lipid class and fatty acid composition of developing eggs and unfed larvae of cod (Gadus morhua). Mar. Biol. 99, 307-313. Holland D. L, and Gabbott P. A. (1971) A micro-analytical scheme for the determination of protein, carbohydrate, lipid and RNA levels in marine invertebrate larvae. J. Mar. Biol. Assoc. U.K. 52, 6594568. Kaitaranta, J. K. and Ackman R. G. (1981) Total lipids and lipid classes of fish roe. Comp. Biochem. Physiol. 69B, 725-729. Love R. M. (1980) The Chemical Biology of Fishes, Vol. H, Advances 1968 1977. Academic Press, London. Mansueti R. (1958) Eggs, larvae and young of the striped bass, Morone saxatilis. Contribution number 112, Maryland Department of Research and Education. Medford B. A. and Mackay W. C. (1978) Protein and lipid content of gonads, liver and muscle of northern pike (Esox lucius) in relation to gonad growth. J. Fish. Res. Bd Can. 35, 213-219. Olsen R. E., Henderson R. J. and Pedersen T. (199l) The influence of dietary lipid classes on the fatty acid composition of small cod Gadus morhua L. juveniles reared in an enclosure in northern Norway. J. exp. Mar. Biol. EcoL 148, 59 76. Ozkizilcik S. and Chu F.-L. E. (1994a) Uptake and metabolism of liposomes by Artemia nauplii. Aquaculture 128, 131-141. Ozkizilcik S. and Chu F.-L. E. (1994b) Evaluation of omega-3 fatty acid enrichment of Artemia nauplii as
food for striped bass Morone saxatilis Walbaum larvae. J. Wrld Aquac. Soc. 25, 147 154. Parker S. J. and Specker J. L. (1990) Salinity and temperature on whole-animal thyroid hormone levels in larval and juvenile striped bass, Morone saxatilis. Fish Physiol. Biochem. 8, 507-514. Pfeiler E. and Luna A. (1984) Changes in biochemical composition and energy utilization during metamorphosis and leptocephalus larvae of the bonefish (Albula). Environ. Biol. Fishes. 10, 243-251. Phleger C. F. and Grigor (1990) Role of wax esters in determining buoyancy in Haplostethus atlanticus (Berysciformes: Trachichthyidae). Mar. Biol. 105, 229 233. Rogers B. A., Westin D. T. and Saila S. B. (1977) Life stage duration studies on Houston River striped bass Morone saxatilis (Walbaum). University of Rhode Island. Marine Technical Report Number 31. Russell F. S. (1976) The Eggs and Planktonic Stages of British Marine Fishes. Academic Press, New York. Sheridan M. A., Allen W. V. and Kerstetter T. H. (1983) Seasonal variations in the lipid composition of the steelhead trout, Salmo gairdneri Richardson, associated with the parr-smolt transformation. J. Fish Biol. 23, 125 134. Sheridan M. A., Allen W. V. and Kerstetter T. H. (1985) Changes in the fatty acid composition of steelhead trout, Salmo gairdneri Richardson, associated with parr-smolt transformation. Comp. Biochem. Physiol. 80B, 671 676. Smith S. (1957) Early development and hatching. In The Physiology of Fishes (Edited by Brown M. E.), Vol. 1, pp. 232-359. Academic Press, New York. Vetter R. D. and Hodson R. E. (1983) Energy metabolism in a rapidly developing imarine fish egg, the red drum (Sciaenops ocellata). Can. J. Fish. Aquat. Sci. 40, 6274534. Vitiello F. and Zanetta J.-P. (1978) Thin layer chromatography of phospholipids. J. Chromatogr. 166, 637-640. Vuorela R., Kaitaranta J. and Linko R. R. (1979) Proximate composition offish roe in relation to maturity. Can. Inst. Food Sci. Technol. J. 12, 186 188. Webster C. D. and Lovell R. T. (1990) Response of striped bass larvae fed brine shrimp from different sources containing different fatty acid composition. Aquaculture 90, 49451. Woo N., Bern H. A, and Nishioka R. S. (1978) Changes in body composition associated with smoltification and premature transfer to seawater in coho salmon (Onchorhynchus kisutch) and king salmon (O. tschawytscha). J. Fish Biol. 13, 421~,28. Youson J. H. (1988) First metamorphosis. In Fish Physiology (Edited by Hoar W. S. and Randall D. J.), Vol. X1, Part B, pp. 135-184. Academic Press, New York.