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The lipid composition of early stage western rock lobster (Panulirus cygnus) phyllosoma: importance of polar lipid and essential fatty acids.

ABSTRACT Total lipid, lipid class, and fatty acid analyses were conducted on fed and starved stage I and II phyllosoma of the western rock lobster Panulirus cygnus. In both stages, the decrease in dry mass of starved larvae and increase in dry mass of Artemia-fed larvae were accompanied by a decrease and increase in lipid content, respectively. Lipid accounted for 6.7% of the decrease in dry mass in starved stage I larvae, which increased to 35.0% in stage II larvae. Also, lipid accounted for 6.2% of the increase in dry mass of fed stage I larvae, increasing to 19.2% in stage II larvae. The major lipid classes in all phyllosoma samples were polar lipids (84.1-94.3%) followed by sterols (6.6-12.1%; mainly cholesterol). Gravimetrically, fed larvae increased predominantly in polar lipid whereas in starved larvae, polar lipid was the major lipid class catabolized, with the sterol content not changing significantly. Hydrocarbons, wax esters, diacylglyceryl ether, triacylglycerols, and free fatty acids were all minor lipid classes (<5% of total lipid). Fatty acid analysis showed six major components present; 16:0, 18:1n-9, 18:0, 20:4n-6 (arachidonic acid; AA), 20:5n-3 (eicosapentaenoic acid; EPA) and 22:6n-3 (docosahexaenoic acid; DHA). These fatty acids all increased gravimetrically in fed larvae and decreased in starved larvae. In starved larvae, small decreases were seen in the relative contribution of EPA, DHA, 16:1n-7, and 18:1n-9, with AA increasing. In fed larvae, most of the major fatty acids remained at a similar relative level, and larvae were able to accumulate AA and EPA, but not DHA, above the relative level (%) in Artemia. The results are useful in the identification of nutrients required during development and as such with the design of diets used in phyllosoma culture.

KEY WORDS: Artemia, fatty acids, lipid, Panulirus cygnus, phyllosoma, polar lipid, rock lobster

INTRODUCTION

With increased product demand and value, aquaculture or enhancement of spiny lobster fisheries is currently receiving considerable interest due to wild fisheries being fully exploited worldwide (Kittaka 1994, Phillips et al. 2000). The western rock lobster, Panulirus cygnus, is found on the lower west coast of Australia and supports the world's largest rock lobster fishery (Phillips et al. 21)00). To ensure sustainability, a major spiny lobster aquaculture industry would need to be based on culture of phyllosoma from hatching through their entire larval development (Crear et al. 1998, Kittaka and Booth 1994). A major hurdle in complete culture of spiny lobsters is the long and complex larval component of its life history. Although the larval life cycle has been completed in the laboratory for a number of species, only limited success has been achieved (e.g., Booth 1995, Kittaka 1997), with many studies experiencing high mortality in early stages (e.g., Kittaka et al. 1997, Kittaka 1988, Kittaka & Ikegami 1988).

Nutrition is regarded as a key factor controlling survival and growth in crustacean larval culture (Mikami et al. 1995). Very little is known about the feeding of phyllosoma in the wild, creating a need for larval nutritional research, as nutritional studies on spiny lobsters (Palinuridae), particularly on the larval stages that are difficult to rear, are scarce (Kanazawa and Koshio 1994, Phleger et al. 2001). Phyllosoma have been reared on Artemia and mussel gonad with varying success (Illingworth et al. 1997, Kittaka 1988), however, appropriate nutrition requires the identification of the essential elements within the diet (Kanazawa and Koshio 1994). Lipid has been found to be of prime importance in crustacean larval stages (Kattner et al. 1994, Sasaki et al. 1986), and appears to he the main storage product in late stage phyllosoma, which is then used as an energy source during the nonfeeding puerulus stage (Jells et al. 1999, Jeffs et al. 2001).

Starvation experiments are one means to determine nutritional requirements of fish and crustacean larvae, with the retention of specific fatty acids (FA) during starvation being interpreted as a requirement for that specific FA (Olsen 1998). Korea et al. (1989) found fish larvae (Sparus aurata) conserved essential n-3 FA during starvation, especially docosahexaenoic acid (DHA) and eicosapeataenoic acid (EPA), and considered this to be a reasonable biochemical strategy as these FA are more valuable as components of membranes than as energy sources. Biochemical studies during starvation and feeding of prawn larvae have also supplied valuable information on the best nutrient specification of food for the larvae (D'Souza 1998).

The objective of this work was to examine which lipids (lipid classes and fatty acids) were of greater nutritional importance for stage I and II P. cygnus phyllosoma by comparing patterns of conservation and loss in starved and fed larvae. The aim was to provide information that would be useful in defining suitable diets with profiles appropriate for phyllosoma culture.

MATERIALS AND METHODS

Broodstock

The study was undertaken at the Western Australian Marine Research Laboratories, Perth, Australia. Broodstock animals were kept in the laboratory under 12L:12D light cycle and led daily with live mussels (Mytilus edulis) and routinely with fish and abalone. Larvae used in the stage I experiment hatched from a lobster (128.3 mm carapace length) mated in the laboratory, with oviposition of eggs (24 May 2001) at 25[degrees]C and incubation in a tank at 22[degrees]C (32-34 ppt), with larvae hatching after 39 days incubation. Larvae used in the stage II experiment hatched from a lobster (105.0 mm carapace length) mated in the laboratory, with oviposition of eggs (29 July 2001) at 25[degrees]C and incubation in a tank at 19[degrees]C (32-34 ppt), with larvae hatching after 57 days incubation.

Experimental System

The system used for rearing the phyllosoma has successfully been used to rear southern rock lobster (Jasus edwardsii) phyllosoma to their final stage (Ritar 2001). Seawater used in the experiments was heated to 23[degrees]C, filtered to 1 [mu]m and UV sterilized (UViVF-9, 30 W) before entering the circular 30-L plastic rearing tubs. Water entered the tubs through tour equally spaced nozzles (jets) positioned close to the bottom perimeter of the tub, with another two toward the bottom center of the tub to provide a circular water flow, thereby keeping phyllosoma moving in the water column. Each tub had a water flow of approximately 1 L/min, and the volume was maintained at 10 L. Excess water exited through screens positioned on the side of the tubs. Phyllosoma were fed daily with Artemia at 3/mL. Every morning, remaining Artemia were removed from the system by replacing the usual screens, "feeding filters" (200 [micro]m), with "cleaning filters" (1500 [micro]m) for approximately h that allowed the Artemia to be flushed out of the tubs. The feeding filters were replaced and freshly enriched Artemia were then added to the tubs. Phyllosoma were transferred to clean tubs weekly. Phyllosoma that were starved during stages I and II received the same daily procedure except they were not fed. Larvae starved in stage II had been fed in stage I. The number of larvae added to the tubs was estimated volumetrically, and larvae were randomly stocked at approximately 1,500/tub.

Artemia

Phyllosoma were fed with Artemia (Great Salt Lake) that had been on-grown for 4-5 days, after reaching instar II, using Algamac 2000 (Biomarine, Aquafauna) and an Isochrysis marine algal concentrate (Reed Mariculture, San Jose, CA). Artemia were also enriched for 18 h (two feedings, 1600 and 0200) with Algamac 2000 prior to feeding to the phyllosoma.

Sampling Protocol

All phyllosoma samples for biochemical analysis were taken in triplicate (i.e., three tubs were used for each sample). For stage I analyses, phyllosoma were sampled at hatch (day 0), fed and starved samples in the middle of stage I (day 6), and a sample of fed larvae after molting at the beginning of stage II (day 15). For stage II analysis, phyllosoma were sampled at hatch (day 0), at the beginning of stage II (day 15), fed and starved samples were taken in the middle of stage II (day 20), and a sample of fed larvae after molting at the beginning of stage III (day 26). Larval stages were measured from the anterior margin of the cephalic shield between the eyestalks to the posterior of the abdomen and staged according to Braine et al. (1979). Samples of enriched Artemia were taken for analysis.

Lipid Analysis

Lipid Extraction

Phyllosoma and Artemia samples were filtered onto 47 mm Whatman GFC filters and washed with 0.5 M ammonium formate. Samples were stored at -80[degrees]C, freeze-dried overnight, and weighed to determine dry mass (DM). Samples were quantitatively extracted overnight using a modified Bligh and Dyer (1959) one-phase methanol:chloroform:water extraction (2:1:0.8 v/v/v). The phases were separated the following day by the addition of chloroform and water to give a final solvent ratio of 1:1:0.9 v/v/v methanol:chloroform:water. The total solvent extract (TSE) was concentrated using a rotary evaporator at 40[degrees]C, blown down to dryness under nitrogen, and weighed to determine the total lipid content. Samples were made up in a known volume of chloroform and stored at 20[degrees]C before analysis.

Lipid Classes

To quantify individual lipid classes (LC), an aliquot of the TSE was analyzed using an Iatroscan MK V TH 10 thin layer chromatography-flame ionization detector (TLC-FID) (Iatron Laboratories, Tokyo, Japan). Samples were applied in duplicate to silica gel SIII chromarods (5-[mu]m particle size) using 1-[mu]L disposable micropipettes. Chromarods were developed in a glass tank lined with pre-extracted filter paper. The primary solvent system used for lipid class separation was hexane:diethyl ether:acetic acid (60:17: 0.1 v/v/v), a mobile phase resolving nonpolar compounds such as wax esters (WE), triacylglycerols (TAG), free fatty acids (FFA), and sterols (ST). A second nonpolar solvent system of hexane:diethyl ether (96:4 v/v) was also used to resolve hydrocarbons (HC) from WE, and TAG from diacylglycerol ether (DAGE). After development, the chromarods were oven-dried and analyzed immediately. The FID was calibrated for each compound class (phosphatidylcholine, cholesterol, cholesteryl ester, oleic acid, squalene, TAG [derived from fish oil], WE [derived from fish oil] and DAGE [derived from shark oil], 0.1-10 [mu]g range), and the peaks were quantified using DAPA software (Kalamunda, Western Australia).

Fatty Acids

An aliquot of the TSE was trans-methylated to produce fatty acid methyl esters (FAME) using methanol:chloroform:conc. hydrochloric acid (10:1:1 v/v/v) at 80[degrees]C for 2 h. The FAME produced were extracted into hexane:chloroform (4:1 v/v. 3 x 1.5 mL), reduced under nitrogen to dryness, and treated with N,O-bis-trimethylsilyl)-trifluoroacetamide (BSTFA, 100 [mu]L, 70[degrees]C, overnight) to convert ST and alcohols to their corresponding TMSi (trimethylsilyl) ethers. Samples were blown down under nitrogen and an internal standard (C19 and C23, 40 mg/g) was added.

Gas chromatographic (GC) analyses were performed with a Hewlett Packard 5890A GC (Avondale, PA) equipped with a HP-5 cross-linked methyl silicone fused silica capillary column (50 m x 0.32 mm i.d.), a FID, a split/splitless injector, and a HP 7673A auto sampler. Helium was used as the carrier gas. Samples were injected in splitless mode at an oven temperature of 50[degrees]C. After 1 min, the over temperature was raised to 150[degrees]C al 30[degrees]C/min, then to 250[degrees]C at 2[degrees]C/min and finally to 300[degrees]C at 5[degrees]C/min. Peaks were quantified with Waters Millennium software (Milford, MA). Individual components were identified using mass spectral data and by comparing the retention time data with those obtained for authentic and laboratory standards. GC results are subject to an error of [+ or -]5% of individual component area. GC-mass spectrometric (GC-MS) analyses were performed on a Finnigan Thermoquest GCQ GC-mass spectrometer (Austin, TX) fitted with an on-column injector. The GC was fitted with a capillary column similar to that described above.

Statistical Analysis

Results were analyzed using one-way ANOVA with Tukey's test used for multiple comparisons. Percentage data was arcsine [square root of] transformed and gravimetric data [square root of] transformed to make the data normal and homogenous. When two samples were compared (hatch sizes), a t-test was used. Statistical analyses were performed using Statistica software (StatSoft Inc., Tulsa, OK, version 6). Data is presented as mean [+ or -] SD, and results were considered significantly different at P [less than or equal to] 0.05.

RESULTS

Larvae from the two batches did not significantly differ in DM or lipid content (mg/g DM and [mu]g/phyllosoma) at hatch; however, the lipid content was slightly elevated in larvae from the second hatch. For stage I samples, changes in fed and starved larvae were compared with newly hatched larvae (from first hatch), and for stage II samples to newly molted stage II larvae (from the second hatch).

Phyllosoma and Lipid Amounts

By mid stage, the DM had increased in stage I and II (significant) fed larvae (16.5 and 33.6%, respectively) and conversely decreased in starved stage I (significant) and II larvae (29.1 and 10.9%, respectively) (Table 1). Larvae also showed increases in dry mass (DM) by the time they molted to the next stage (Table 1). As with DM, the lipid content (mg/g DM and [mu]g/phyllosoma) of stage I and II larvae increased in fed samples and decreased in starved samples (Table 1). In stage I larvae, the increase in lipid content of fed larvae accounted for 6.2% of the increase in DM whereas the lipid decrease in starved larvae accounted fur 6.7% of the decrease in DM. In stage II larvae, lipid accounted for a much greater percentage of the increase in DM of fed larvae, 19.2%, and also accounted for a much larger portion of the decrease in starved stage II larvae, 35.0%. Although newly molted stage III did have a similar amount of lipid/phyllosoma compared with fed stage II larvae, total lipid (nag/g DM) decreased significantly after the molt (Table 1).

Lipid Class

In all phyllosoma samples, polar lipid (PL) was the major LC (82.0-94.3%) (Table 2). As with lipid amounts, PL concentration in larvae differed from the different hatches, larvae from the second hatch (49.1 mg/g DM) having significantly increased levels compared with larvae from the first hatch (37.6 mg/g DM) (Table 2, Fig. 1). PL was also the major LC used during starvation and on an absolute basis was significantly reduced in starved stage I and II larvae (Table 2, Fig. 1). The amount of PL in starved stage I larvae (27.2 mg/g DM) was 27.6% less than in newly hatched larvae (37.6 mg/g DM), and increased by 8.4% in led phyllosoma (40.8 mg/g DM) (Fig. 1). For stage II larvae, the amount of PL in starved larvae (29.1 mg/g DM) was 53.9% less than in newly molted stage II larvae (63.0 mg/g DM), and increased by 50.2% in fed stage II larvae (94.7 mg/g DM) (Fig. 1). In both stages, the increase in PL (mg/g DM) almost solely accounted for the increase in lipid (mg/g DM). ST were the next most abundant lipid class (largely cholesterol) comprising between 5.1-12.1% of the total lipid (Table 2). In relative proportions, ST levels (%) increased in starved larvae (significant in stage II), with no significant decrease in amount (mg/g DM) (Table 2, Fig. 1). HC were the next most abundant LC (0.4-4.6%), followed by WE (0.1-2.3%), FFA (0.1-1.1%), DAGE and TAG ([less than or equal to] 0.2%) (Table 2).

[FIGURE 1 OMITTED]

Enriched Artemia contained high relative and absolute levels of PL (62.0%; 83.9 mg/g DM), although unlike the phyllosoma samples, also bad high levels of TAG (31.0%; 41.8 mg/g DM). This was followed by ST (3.3%; 4.5 mg/g DM), and FFA (3.1%; 4.2 mg/g DM), with HC, DAGE. and WE being minor components ([less than or equal to] 0.3%; <0.5 mg/g DM) (Table 2, Fig. 1).

Fatty Acid

Gravimetrically, newly hatched larvae from the second hatch were significantly elevated in total FA and all the major FA compared with the first hatch (Tables 3 and 4, Fig. 2). Of the 55 fatty acids (FA) identified, 18 individual FA had some concentrations [greater than or equal to] 1% in the phyllosoma samples. The six most abundant FA ([greater than or equal to] 5%) were 20:5n-3 (EPA, 13.7-21.3%), 16:0 (palmitic acid, 10.7-15.2%), 18:1n-9c (oleic acid, 10.6-14.6%), 22:6n-3 (DHA, 7.1-13.0%), 20:4n-6 (arachidonic acid, AA, 5.8-12.4%), and 18:0 (stearic acid, 8.5-13.9%) (Tables 3 and 4). These six components generally accounted for between 68.6 to 76.9% of the total FA.

[FIGURE 2 OMITTED]

The major FA in fed stage I larvae showed similar relative contributions to newly hatched larvae, except there was a significant increase in EPA and decrease in 16:0 (Table 3). Similarly, fed stage II larvae showed a similar relative contribution of the major FA compared with newly molted stage II larvae, although 16:0 was significantly elevated (Table 4). Gravimetrically, total FA (mg/g DM) increased significantly in fed stage I and II larvae as did the major FA (Fig. 2). The sum of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) in led stage I and II larvae did not change. The sum of polyunsaturated fatty acids (PUFA) did not significantly change in fed stage I and II larvae, however there was a small increase (Table 3 and 4). Both stage I and II fed larvae showed an increase in n-3/n-6 ratio (significant in stage I) (Tables 3 and 4). The EPA/AA ratio showed a significant increase in both fed stage I and II larvae, largely due to an increase in EPA for stage I, and a decrease in AA, particularly for stage II larvae (Tables 3 and 4). The DHA/EPA ratio did not significantly change in fed stage I or II larvae.

Starved stage I and II larvae showed a gravimetric decrease in total FA, although this was only significant in stage II larvae (Tables 3 and 4). Individually, most of the major FA decreased in starved stage I larvae, although only DHA decreased significantly, and 18:0 actually increased (Fig. 2). In starved stage II larvae, all the major FA significantly decreased gravimetrically. On a percentage basis, AA increased in starved stage I and II larvae (significant in stage II), with no significant change in EPA and DHA (Table 3 and 4). Starved stage I larvae showed no significant difference in the sum of MUFA or PUFA compared with newly hatched larvae, however SFA significantly increased (Table 3). Starved stage II larvae did not show a difference in the sum of SFA, however PUFA significantly increased and MUFA significantly decreased (Table 4). In stage I and II starved larvae, there was no change in the n-3/n-6 ratio (Tables 3 and 4). A reduction in the EPA/AA ratio was observed in starved larvae (not significant), with the DHA/EPA ratio not changing (Tables 3 and 4).

The total FA content significantly decreased in newly molted stage III larvae compared with fed stage II larvae. However, newly molted stage II larvae actually contained a higher total FA content than led stage I larvae (Table 3 and 4).

The percentage FA composition of the Artemia was dominated by 16:0 (19.7%), followed by DHA (14.3%), 18:1n-9c (12.3%), EPA (10.7%), 18:1n-7c (9.8%), and 16:1n-7c (6.2%). Other FA were present at [less than or equal to] 6%, with AA at 2.3% (Table 3). Phyllosoma were able to accumulate AA and EPA at percentage levels above that in Artemia, but not DHA, which was always lower in phyllosoma.

DISCUSSION

Changes in Phyllosoma Dry Mass and Lipid Content

In both larval stages, there were increases in the DM and in the amount of lipid in fed larvae, with decreases in starved larvae. Changes were more significant in stage II larvae (Table 1). Lipids are used during starvation in other crustaceans (Virtue et al. 1993), and previous research has found lipid a key component in larval stages of spiny lobsters (Jeffs et al. 1999, Phleger et al. 2001). However, lipid only accounted for 6.5% of the decrease in DM in starved stage I larvae whereas this increased to 35.0% for stage II larvae. Also, lipid only accounted for 6.2% of the increase in DM of fed stage I larvae, increasing to 19.7% in stage II larvae. This indicates that lipid was not the major nutrient catabolized during starvation or accumulated by the feeding phyllosoma. Smith et al. (2003) suggested that protein, and to a lesser extent carbohydrate, would have been preferentially catabolized during starvation of stage I J. edwardsii phyllosoma in which lipid accounted for 17.8% in the loss of DM. This higher value in J. edwardsii may be related to the larger size of stage I larvae compared with P. cygnus. D'Souza (1998) found starved Penaeus japonicus and Penaeus monodon larvae exhibited gravimetric decreases in both lipid and protein.

Larvae molting to successive stages showed an increase in DM, with stage II larvae molting to stage III also showing a reduction in the lipid content (Table 1), possibly used during the molt. Smith et al. (2003) suggested that the increase in lipid accumulation seen in fed J. edwardsii phyllosoma might be used during the subsequent moult, as found with American lobster (Homarus americanus) larvae (Sasaki 1984). A decrease was not seen in newly molted stage II P. cygnus larvae compared with fed stage I, the lipid level was actually higher in the newly molted stage II larvae (Table 1). This may be attributable to a large variation in the molting period of the larvae. Larvae from the first hatch took 3 days to all molt to stage II before a sample was taken. Therefore, many of the sampled larvae may have started to accumulate lipid.

Lipid Class

Variation Between Hatches and Species

Newly hatched larvae from the second hatch had elevated concentrations of lipid (PL and total FA) compared with larvae from the first hatch, with larvae from the second hatch also being significantly larger (Tables 1, 2, 3, and 4). This may be attributable to different temperatures during egg incubation. Both females extruded eggs at 25[degrees]C, however eggs from the first hatch were incubated in water at 22[degrees]C whereas those from the second hatch were al 19[degrees]C. Smith et al. (2002) found newly hatched J. edwardsii phyllosoma from warmer water were significantly smaller. The warmer incubation temperature may have resulted in more energy being used for metabolism and less for development of the embryo. This difference at hatching between batches of P. cygnus larvae had become significant at the molt to stage II (Table 1). Larvae from the second hatch showed significantly higher lipid content (PL and total FA), suggesting the condition of larvae at hatch affected their condition into stage II, and possibly for further culture.

The absence or very low levels of TAG in phyllosoma samples has been a common feature found in studies so far (Nelson et al. 2003, Phleger et al. 2001), including this study (Table 2). TAG is generally the most common storage lipid in animals, used as a short-term energy reserve, and it is generally catabolized primarily during starvation (Koven et al. 1989, Olsen 1998, Phleger et al. 2001). This is in contrast to PL, which plays an important structural role and is usually preferentially conserved (Koven et al. 1989). PL was the major LC in P. cygnus larvae comprising 82.0-94.3% of the total lipid from all samples (Table 2), as found with J. edwardsii phyllosoma (Nelson et al. 2003, Phleger et al. 2001). In this study, the increase found in lipid (mg/g DM) in both stages of fed P. cygnus larvae corresponded almost completely to the increase in PL (mg/g DM). The use of PL as the dominant storage medium in the puerulus stage of rock lobsters is unlike many other marine taxa, including crustacea, which tend to use TAG and WE (Jeffs et al. 2001, Sasaki 1984). Jeffs et al. (2001) found PL reserves were primarily used during the puerulus stage of J. edwardsii and suggested the use of PL as a storage medium in the puerulus may be related to its characteristic transparency, an important feature of this nektonic stage that is highly vulnerable to pelagic visual predators. Jeffs et al. (2001) suggested pueruli avoid storing neutral lipid because of its opaque nature. The prevalence of PL as the major LC in phyllosoma larvae may also be due to their transparency, which could provide protection during their long larval ocean phase.

PL was also the major LC used during starvation and was significantly reduced in starved larvae from both stages (Table 2, Fig. 1). Lipid was found to be the primary energy source used during the nonfeeding puerulus stage and PL was also the major LC used (Jeffs et al. 1999, Jeffs et al. 2001). PL was also the only LC to be depleted when stage II larvae molted to stage III, with a significant gravimetric reduction (Fig. 1).

ST (mainly cholesterol) was the next most abundant LC in the phyllosoma samples (Table 2). PL, together with cholesterol and sphingolipids, are omnipresent components of cell membranes and are therefore both structurally and functionally important (Olsen, 1998). Unlike PL, relative amounts of ST increased in starved larvae, with no significant decrease in gravimetric amount (mg/g DW) (Table 2, Fig. 1). This retention may indicate the importance of ST, as ST are used not only in membranes but also in the transport of lipids (Ackman 1998).

Phyllosoma were able to maintain a higher percentage of PL and ST than found in their diet (Table 2). Artemia contained lower levels of PL (62.0%) and ST (3.3%), with higher levels of TAG (31.0%) and FFA (3.1%) than phyllosoma (Table 2). It appears PL and ST are important nutrients to the phyllosoma, hence their accumulation at levels (%) above that found in their food. However, TAG is almost absent (<0.2%) in the phyllosoma samples (Table 2).

The phyllosoma lipid was around 90% PL, and comparatively the PL in Artemia was much lower (60%). TAG makes up a large portion in Artemia, and it is possible that phyllosoma cannot absorb or accumulate the TAG as efficiently as PL to gain nutrients.

Fatty Acids

The major FA and their profiles in newly hatched P. cygnus larvae (Tables 3 and 4) are similar to those reported for newly hatched J. edwardsii phyllosoma (14.2-14.6% 16:0, 12.6-13.3% 18:1n-9c, 6.9-7.1% 18:0, 11.6-11.9% AA, 14.8-15.3% EPA, and 7.6-8.1% DHA; Phleger et al. 2001). Fed P. cygnus larvae also showed a similar relative contribution from the major FA, with all the major FA significantly increasing gravimetrically. However, the relative levels were different to those found in their diet. The FA distribution of animals is believed to be primarily determined by the composition of their dietary FA (Napolitano 1998, Olsen 1998). In comparison, for the current study 16:0 and DHA were at lower relative levels than found in the Artemia, with 18:0, AA, and EPA at levels above that found in the Artemia, possibly indicating a preferential accumulation of these FA. DHA showed a continual reduction in relative contribution as the phyllosoma developed (fed and molting samples). This may suggest that the DHA was not in a form that the phyllosoma could absorb and use or was not at high enough levels. Nichols et al. (2001) examined a wide range of potential prey items for J. edwardsii phyllosoma and found that DHA was in average 26%, markedly higher than that in the Artemia. The prey also contained on average 77% PL, again far greater than occurring in Artemia. AA also declined throughout development; however the level of AA in the Artemia was always much lower than found in the phyllosoma.

The increase in the EPA/AA ratio in fed larvae suggests a higher accumulation of EPA in fed larvae, however, starved larvae did exhibit a reduced ratio (not significant) suggesting more AA was conserved during starvation (Table 3 and 4). The DHA/EPA ratio did not show major changes in fed or starved stage I or II larvae, although some where statistically significant, suggesting both FA were used/retained at a similar rate (Tables 3 and 4).

To formulate a diet that meets the essential fatty acid (EFA) requirements of a given species, it is necessary to know the dominant EFA series for this species (Corraze 2001). Experiments have found that during starvation larvae conserve important FA, with starvation suggested as one way to determine nutritional requirements of larvae (Koven et al. 1989. Olsen 1998). Starved P. cygnus larvae showed a gravimetric decrease in total FA, however, a number of individual FA showed no change or assumed a greater ranking in the FA profile, possibly signifying a greater degree of importance. The FA changes were however dependent on the stage tested. The relative contribution of AA increased in starved stage I and II larvae, although the change in starved stage I larvae was not significant. EPA and DHA showed increases in stage II larvae, however, in starved stage I larvae there was a small decrease. D'Souza (1998) found PUFA (AA, EPA and DHA) were conserved in starved Penaeus larvae and suggested this was most likely due to their primarily structural role.

Starved stage I larvae showed a significant increase in SFA, with no change in MUFA or PUFA. However, in starved stage II larvae, MUFA significantly decreased whereas PUFA significantly increased, possibly indicating a preferential use of MUFA during starvation and a sparing or retention of PUFA. Kattner et al. (1994) found the FA profile of larval caridean shrimp also showed major differences dependent on stage and larval sample. The variation in results in the present study suggest a response for P. cygnus which is dependent on the stage of the larvae.

CONCLUSIONS

Although lipid was not a major component of the larvae and was not the major nutrient accumulated in fed larvae or used in starved larvae, lipid became a greater component in stage II larvae compared with stage I larvae, suggesting that lipid likely assumes a greater importance as larvae progress through developmental stages. Further studies should also assess the importance of other nutrients (i.e., protein and carbohydrate) in phyllosoma development. The LC profile of the phyllosoma samples was dominated by PL. PL was also the main LC used during starvation and accounted for almost the entire increase in lipid in fed larvae. ST was the next most abundant LC and showed an increase in relative contribution in starved larvae, its retention possibly related to its important structural/function role. Artemia had high levels of TAG, however phyllosoma never accumulated TAG. The FA changes occurring in larvae appeared to be stage dependent, however PUFA, such as AA, EPA and DHA, were generally conserved (particularly in stage II larvae). Results from the current study provide information that will assist in formulating diets for phyllosoma culture. Larval feeds containing lipids rich in EFA (EPA, DHA and AA) and in a form allowing them to synthesize PL may be more suitable for future research with P. cygnus. The Artemia, as enriched in this study, do not meet these criteria, with current work to focus on enriching Artemia to make them reflect the perceived profile.
TABLE 1.
The dry mass (DM) per individual (mg), lipid content (mg/g
DM and [micro]g/individual) and size (mean [+ or -] SD) of
phyllosoma larvae of the western rock lobster, Panulirus
cygnus, during feeding and starvation.

 Mass/Ind (mg) *

Hatch (1) 0.079 [+ or -] [0.004.sup.a]
Stage I middle fed 0.092 [+ or -] [0.007.sup.ab]
Stage I middle starved 0.056 [+ or -] [0.004.sup.f]
Stage II beginning 0.099 [+ or -] [0.001.sup.bc]
Hatch (2) 0.081 [+ or -] [0.006.sup.a]
Stage II beginning 0.110 [+ or -] [0.004.sup.c]
Stage II middle fed 0.147 [+ or -] [0.007.sup.d]
Stage II middle starved 0.098 [+ or -] [0.004.sup.bc]
Stage III beginning 0.222 [+ or -] [0.015.sup.e]

 Lipid (mg/g DM) *

Hatch (1) 42.9 [+ or -] [1.2.sup.ab]
Stage I middle fed 45.5 [+ or -] [1.4.sup.ab]
Stage I middle starved 33.3 [+ or -] [4.3.sup.b]
Stage II beginning 48.0 [+ or -] [5.8.sup.a]
Hatch (2) 53.5 [+ or -] [5.7.sup.ac]
Stage II beginning 68.9 [+ or -] [7.2.sup.c]
Stage II middle fed 100.4 [+ or -] [6.8.sup.d]
Stage II middle starved 34.6 [+ or -] [4.5.sup.b]
Stage III beginning 66.0 [+ or -] [5.9.sup.c]

 Lipid ([micro]g/ind) *

Hatch (1) 3.4 [+ or -] [0.1.sup.a]
Stage I middle fed 4.2 [+ or -] [0.2.sup.ab]
Stage I middle starved 1.9 [+ or -] [0.1.sup.e]
Stage II beginning 4.8 [+ or -] [0.5.sup.b]
Hatch (2) 4.3 [+ or -] [0.2.sup.ab]
Stage II beginning 7.6 [+ or -] [0.9.sup.c]
Stage II middle fed 14.8 [+ or -] [0.4.sup.d]
Stage II middle starved 3.4 [+ or -] [0.4.sup.a]
Stage III beginning 14.6 [+ or -] [0.4.sup.d]

 Size (mm) ([dagger])

Hatch (1) 1.76 [+ or-] [0.02.sup.a]
Stage I middle fed
Stage I middle starved
Stage II beginning
Hatch (2) 1.81 [+ or -] [0.02.sup.b]
Stage II beginning
Stage II middle fed
Stage II middle starved
Stage III beginning

Hatch 1 and 2 are from different females. Data, within a
column, not sharing a common superscript are significantly
different (P < 0.05).

* n = 3.

([dagger]) n = 20; measured horn the anterior margin of the
cephalic shield to the posterior of the abdomen.

TABLE 2.
Lipid class composition (mean [+ or -] SD; n = 3) as
percentage of total lipid in western rock lobster
(Panulirus cygnus) phyllosoma and enriched Artemia.

 Sample Hydrocarbons

Stage I--hatch 2.5 [+ or -] [1.2.sup.abc]
Stage I--middle fed 1.6 [+ or -] [0.2.sup.acd]
Stage I--middle starved 4.6 [+ or -] [1.4.sup.b]
Stage II--beginning 2.9 [+ or -] [0.6.sup.ab]

Stage I--hatch 0.7 [+ or -] [0.2.sup.cd]
Stage II--beginning 1.1 [+ or -] [0.8.sup.acd]
Stage II--middle fed 0.4 [+ or -] [0.2.sup.d]
Stage II--middle starved 2.6 [+ or -] [1.2.sup.abc]
Stage III--beginning 0.7 [+ or -] [0.4.sup.cd]

Artemia 0.3 [+ or -] 0.0

 Sample Wax Esters

Stage I--hatch 1.3 [+ or -] [0.5.sup.ab]
Stage I--middle fed 0.3 [+ or -] [0.1.sup.bcd]
Stage I--middle starved 2.3 [+ or -] [0.7.sup.a]
Stage II--beginning 1.0 [+ or -] [0.3.sup.ad]

Stage I--hatch 0.1 [+ or -] [0.1.sup.c]
Stage II--beginning 0.6 [+ or -] [0.6.sup.bcd]
Stage II--middle fed 0.1 [+ or -] [0.0.sup.c]
Stage II--middle starved 0.6 [+ or -] [0.4.sup.bcd]
Stage III--beginning 0.2 [+ or -] [0.1.sup.cd]

Artemia 0.1 [+ or -] 0.0

 Sample DAGE

Stage I--hatch 0.1 [+ or -] 0.0
Stage I--middle fed 0.1 [+ or -] 0.1
Stage I--middle starved 0.1 [+ or -] 0.1
Stage II--beginning 0.0 [+ or -] 0.0

Stage I--hatch 0.0 [+ or -] 0.0
Stage II--beginning 0.1 [+ or -] 0.0
Stage II--middle fed 0.0 [+ or -] 0.0
Stage II--middle starved 0.1 [+ or -] 0.0
Stage III--beginning 0.1 [+ or -] 0.0

Artemia 0.2 [+ or -] 0.1

 Sample TAG

Stage I--hatch 0.1 [+ or -] [0.0.sup.ab]
Stage I--middle fed 0.1 [+ or -] [0.0.sup.ab]
Stage I--middle starved 0.2 [+ or -] [0.1.sup.b]
Stage II--beginning 0.2 [+ or -] [0.1.sup.ab]

Stage I--hatch 0.1 [+ or -] [0.0.sup.ab]
Stage II--beginning 0.1 [+ or -] [0.0.sup.ab]
Stage II--middle fed 0.0 [+ or -] [0.0.sup.a]
Stage II--middle starved 0.1 [+ or -] [0.0.sup.ab]
Stage III--beginning 0.1 [+ or -] [0.0.sup.ab]

Artemia 31.0 [+ or -] 2.7

 Sample Free Fatty Acids

Stage I--hatch 0.5 [+ or -] [0.1.sup.ab]
Stage I--middle fed 0.2 [+ or -] [0.1.sup.a]
Stage I--middle starved 1.1 [+ or -] [0.6.sup.b]
Stage II--beginning 0.4 [+ or -] [0.4.sup.a]

Stage I--hatch 0.1 [+ or -] [0.0.sup.a]
Stage II--beginning 0.3 [+ or -] [0.2.sup.a]
Stage II--middle fed 0.1 [+ or -] [0.0.sup.a]
Stage II--middle starved 0.5 [+ or -] [0.1.sup.ab]
Stage III--beginning 0.1 [+ or -] [0.1.sup.a]

Artemia 3.1 [+ or -] 0.2

 Sample Sterols

Stage I--hatch 7.7 [+ or -] [0.6.sup.ab]
Stage I--middle fed 8.2 [+ or -] [0.4.sup.ab]
Stage I--middle starved 9.8 [+ or -] [0.7.sup.ad]
Stage II--beginning 7.3 [+ or -] [1.4.sup.bc]
Stage I--hatch 7.2 [+ or -] [0.6.sup.bc]
Stage II--beginning 6.6 [+ or -] [0.9.sup.bc]
Stage II--middle fed 5.1 [+ or -] [1.0.sup.c]
Stage II--middle starved 12.1 [+ or -] [0.6.sup.d]
Stage III--beginning 7.1 [+ or -] [0.9.sup.bc]

Artemia 3.3 [+ or -] 0.3

 Sample Polar Lipids

Stage I--hatch 87.8 [+ or -] [1.4.sup.ad]
Stage I--middle fed 89.6 [+ or -] [0.4.sup.ab]
Stage I--middle starved 82.0 [+ or -] [2.7.sup.e]
Stage II--beginning 88.3 [+ or -] [0.6.sup.abd]
Stage I--hatch 91.8 [+ or -] [0.3.sup.bc]
Stage II--beginning 91.4 [+ or -] [2.3.sup.abc]
Stage II--middle fed 94.3 [+ or -] [0.7.sup.c]
Stage II--middle starved 84.1 [+ or -] [1.3.sup.de]
Stage III--beginning 91.8 [+ or -] [1.2.sup.ab]

Artemia 62.0 [+ or -] 2.6

Data, within a column, not sharing a common superscript are
significantly different ([alpha] = 0.05).

TAG, triacylglycerol; DAGE, diacylglycerols ether.

TABLE 3.

Percentage fatty acid (FA) composition and total FA levels (mg/g DM)
at hatch, after feeding, starvation, and molting (mean [+ or -] SD;
n = 3) of western rock lobster (Panulirus cygnus) phyllosoma and
enriched Artemia.

 Hatch I

 Stage I

 Fatty Acid Hatch (1)

14:0 0.9 [+ or -] [0.1.sup.ab]
16:1n-7c 2.8 [+ or -] [0.2.sup.ac]
16:0 12.2 [+ or -] [0.8.sup.a]
17:0 0.8 [+ or -] [0.1.sup.a]
18:2n-6 1.1 [+ or -] [0.2.sup.a]
18:1n-9c 11.2 [+ or -] [0.6.sup.ad]
18:1n-7c 3.4 [+ or -] [0.4.sup.a]
18:0 8.5 [+ or -] [0.2.sup.a]
18:0 FAde 2.8 [+ or -] [0.2.sup.a]
20:4-6 AA 11.2 [+ or -] [1.0.sup.ae]
20:5n-3 EPA 16.7 [+ or -] [1.1.sup.a]
20:2n-6 0.8 [+ or -] [0.1.sup.a]
20:1 (n-9/11)c 2.1 [+ or -] [0.3.sup.af]
20:0 0.5 [+ or -] [0.1.sup.a]
22:5n-6 0.4 [+ or -] [0.1.sup.a]
22:6n-3 DHA 11.5 [+ or -] [0.8.sup.ac]
22:5n-3 0.7 [+ or -] [0.1.sup.a]
22:0 0.5 [+ or -] [0.1.sup.a]
Other 8.5 [+ or -] 1.0

Sum SFA 24.8 [+ or -] [1.0.sup.a]
Sum MUFA 21.7 [+ or -] [0.7.sup.ad]
Sum PUFA 45.3 [+ or -] [1.9.sup.ac]
Sum n-3 29.3 [+ or -] [1.8.sup.a]
Sum n-6 14.9 [+ or -] [0.6.sup.ab]
Ratio (n-3)/(n-6) 2.0 [+ or -] [0.2.sup.ab]
Ratio EPA/AA 1.5 [+ or -] [0.1.sup.acd]
Ratio DHA/EPA 0.7 [+ or -] [0.0.sup.a]

Total FA (mg/g DM) 14.8 [+ or -] [0.4.sup.a]

 Hatch I

 Stage I

 Fatty Acid Fed

14:0 0.6 [+ or -] [0.0.sup.bd]
16:1n-7c 1.6 [+ or -] [0.1.sup.b]
16:0 10.7 [+ or -] [0.4.sup.b]
17:0 0.9 [+ or -] [0.0.sup.bd]
18:2n-6 1.4 [+ or -] [0.0.sup.ac]
18:1n-9c 11.2 [+ or -] [0.3.sup.ad]
18:1n-7c 5.3 [+ or -] [0.1.sup.b]
18:0 9.9 [+ or -] [0.2.sup.b]
18:0 FAde 2.0 [+ or -] [0.2.sup.b]
20:4-6 AA 10.8 [+ or -] [0.9.sup.abe]
20:5n-3 EPA 21.3 [+ or -] [1.0.sup.b]
20:2n-6 0.7 [+ or -] [0.0.sup.a]
20:1 (n-9/11)c 1.3 [+ or -] [0.0.sup.b]
20:0 0.8 [+ or -] [0.0.sup.bc]
22:5n-6 0.9 [+ or -] [0.1.sup.b]
22:6n-3 DHA 13.0 [+ or -] [0.4.sup.a]
22:5n-3 0.3 [+ or -] [0.0.sup.b]
22:0 0.9 [+ or -] [0.0.sup.b]
Other 6.2 [+ or -] 0.6

Sum SFA 24.8 [+ or -] [0.7.sup.a]
Sum MUFA 20.8 [+ or -] [0.7.sup.ad]
Sum PUFA 50.4 [+ or -] [1.9.sup.a]
Sum n-3 35.2 [+ or -] [1.2.sup.b]
Sum n-6 14.5 [+ or -] [0.4.sup.ab]
Ratio (n-3)/(n-6) 2.4 [+ or -] [0.3.sup.c]
Ratio EPA/AA 2.0 [+ or -] [0.3.sup.b]
Ratio DHA/EPA 0.6 [+ or -] [0.0.sup.b]

Total FA (mg/g DM) 21.6 [+ or -] [2.5.sup.b]

 Hatch I

 Stage I

 Fatty Acid Starved

14:0 1.0 [+ or -] [0.3.sup.ab]
16:1n-7c 2.2 [+ or -] [0.4.sup.fg]
16:0 12.1 [+ or -] [0.2.sup.a]
17:0 1.1 [+ or -] [0.0.sup.c]
18:2n-6 1.3 [+ or -] [0.2.sup.ac]
18:1n-9c 10.7 [+ or -] [1.0.sup.ad]
18:1n-7c 3.7 [+ or -] [0.2.sup.a]
18:0 12.3 [+ or -] [0.5.sup.cd]
18:0 FAde 3.7 [+ or -] [0.2.sup.d]
20:4-6 AA 12.4 [+ or -] [0.8.sup.e]
20:5n-3 EPA 15.6 [+ or -] [0.7.sup.ac]
20:2n-6 1.2 [+ or -] [0.0.sup.c]
20:1 (n-9/11)c 1.8 [+ or -] [0.1.sup.f]
20:0 1.4 [+ or -] [0.1.sup.e]
22:5n-6 0.2 [+ or -] [0.0.sup.e]
22:6n-3 DHA 10.3 [+ or -] [0.5.sup.ce]
22:5n-3 0.3 [+ or -] [0.1.sup.b]
22:0 1.3 [+ or -] [0.1.sup.c]
Other 8.0 [+ or -] 0.5

Sum SFA 30.5 [+ or -] [0.3.sup.cd]
Sum MUFA 19.7 [+ or -] [1.6.sup.d]
Sum PUFA 43.6 [+ or -] [1.7.sup.c]
Sum n-3 26.8 [+ or -] [1.3.sup.ac]
Sum n-6 15.8 [+ or -] [0.5.sup.a]
Ratio (n-3)/(n-6) 1.7 [+ or -] [0.1.sup.a]
Ratio EPA/AA 1.3 [+ or -] [0.1.sup.d]
Ratio DHA/EPA 0.7 [+ or -] [0.0.sup.ab]

Total FA (mg/g DM) 13.1 [+ or -] [1.6.sup.a]

 Hatch I

 Stage II

 Fatty Acid Beginning

14:0 1.2 [+ or -] [0.1.sup.ac]
16:1n-7c 2.6 [+ or -] [0.1.sup.af]
16:0 13.9 [+ or -] [0.5.sup.c]
17:0 1.1 [+ or -] [0.0.sup.c]
18:2n-6 1.9 [+ or -] [0.1.sup.b]
18:1n-9c 14.6 [+ or -] [0.3.sup.b]
18:1n-7c 9.1 [+ or -] [0.2.sup.c]
18:0 11.9 [+ or -] [0.7.sup.c]
18:0 FAde 1.6 [+ or -] [0.3.sup.c]
20:4-6 AA 8.7 [+ or -] [0.2.sup.bc]
20:5n-3 EPA 14.9 [+ or -] [0.0.sup.ac]
20:2n-6 0.5 [+ or -] [0.0.sup.b]
20:1 (n-9/11)c 1.1 [+ or -] [0.0.sup.bc]
20:0 0.9 [+ or -] [0.0.sup.b]
22:5n-6 1.4 [+ or -] [0.1.sup.cf]
22:6n-3 DHA 7.1 [+ or -] [0.2.sup.bd]
22:5n-3 0.2 [+ or -] [0.0.sup.c]
22:0 1.2 [+ or -] [0.0.sup.cd]
Other 6.6 [+ or -] 0.6

Sum SFA 31.7 [+ or -] [0.9.sup.bc]
Sum MUFA 28.9 [+ or -] [0.3.sup.b]
Sum PUFA 36.5 [+ or -] [0.4.sup.bde]
Sum n-3 23.0 [+ or -] [0.3.sup.cd]
Sum n-6 13.1 [+ or -] [0.1.sup.bc]
Ratio (n-3)/(n-6) 1.8 [+ or -] [0.0.sup.a]
Ratio EPA/AA 1.7 [+ or -] [0.0.sup.ab]
Ratio DHA/EPA 0.5 [+ or -] [0.0.sup.c]

Total FA (mg/g DM) 23.6 [+ or -] [1.8.sup.b]

 Hatch I

 Fatty Acid Artemia

14:0 5.1 [+ or -] 0.0
16:1n-7c 6.2 [+ or -] 0.1
16:0 19.7 [+ or -] 0.2
17:0 0.7 [+ or -] 0.0
18:2n-6 1.7 [+ or -] 0.0
18:1n-9c 12.3 [+ or -] 0.1
18:1n-7c 9.8 [+ or -] 0.1
18:0 5.3 [+ or -] 0.1
18:0 FAde 0.0 [+ or -] 0.0
20:4-6 AA 2.3 [+ or -] 0.0
20:5n-3 EPA 10.7 [+ or -] 0.1
20:2n-6 0.1 [+ or -] 0.0
20:1 (n-9/11)c 0.3 [+ or -] 0.0
20:0 0.2 [+ or -] 0.0
22:5n-6 4.7 [+ or -] 0.2
22:6n-3 DHA 14.3 [+ or -] 0.7
22:5n-3 0.2 [+ or -] 0.0
22:0 0.7 [+ or -] 0.0
Other 6.1 [+ or -] 0.3

Sum SFA 34.0 [+ or -] 0.3
Sum MUFA 30.3 [+ or -] 0.3
Sum PUFA 48.1 [+ or -] 0.5
Sum n-3 38.4 [+ or -] 0.4
Sum n-6 9.3 [+ or -] 0.1
Ratio (n-3)/(n-6) 4.1 [+ or -] 0.0
Ratio EPA/AA 4.6 [+ or -] 0.0
Ratio DHA/EPA 1.3 [+ or -] 0.1

Total FA (mg/g DM) 78.1 [+ or -] 2.3

Data, within a row, not sharing a common superscript are significantly
different (P < 0.05).

Other <1%: 12:0, i14:0, 14:1, C14PUFA, i15:0, a15:0, 15:1, 15:0,
i16:0, C16PUFA, 16:1n-9c, 16:2, 16:1n-5c, 16:0 Fade, br17:1, i17:0,
a17:0, 17:1, 18:3n-6, i18:0, 18:4n-3, 18:3n-3, 18:1n-5c, C19PUFA,
i19:0, 19:1, 20:2. 20:3n-6, 20:2NMI, 20:4n-3 20:1n-7c, C20Falde,
C21PUFA, 21:0, 22:4n-6, 22:1, C28PUFA.

AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic
acid; SFA, saturated fatty acids: MUFA, monounsaturated fatty acids:
PUFA, polyunsaturated fatty acids: NMI, nun-methylene interrupted;
Fade, fatty aldehyde; DM, dry mass.

TABLE 4.
Percentage fatty acid (FA) composition and total FA levels
(mg/g DM) at hatch, after feeding, starvation, and molting
(mean [+ or -] SD; n = 3) of western rock lobster
(Panulirus cygnus) phyllosoma.

 Hatch 2

 Stage I

 Fatty acid Hatch (2)

14:0 0.4 [+ or -] [0.0.sup.d]
16:1n-7c 2.7 [+ or -] [0.0.sup.a]
16:0 13.7 [+ or -] [0.0.sup.c]
17:0 1.2 [+ or -] [0.0.sup.c]
18:2n-6 1.2 [+ or -] [0.0.sup.ac]
18:1n-9c 12.4 [+ or -] [0.0.sup.ac]
18:1n-7c 4.5 [+ or -] [0.0.sup.d]
18:0 9.8 [+ or -] [0.1.sup.ab]
18:0 Fade 3.8 [+ or -] [0.0.sup.d]
20:4n-6 AA 9.9 [+ or -] [0.0.sup.ab]
20:5n-3 EPA 13.7 [+ or -] [0.1.sup.c]
20:2n-6 1.3 [+ or -] [0.0.sup.c]
20:1(n-9/11)c 2.3 [+ or -] [0.0.sup.a]
20:0 0.7 [+ or -] [0.0.sup.bc]
22:5n-6 0.4 [+ or -] [0.0.sup.a]
22:6n-3 DHA 10.5 [+ or -] [0.1.sup.c]
22:5n-3 1.1 [+ or -] [0.0.sup.d]
22:0 0.9 [+ or -] [0.0.sup.b]
Other 10.1 [+ or -] 0.0

Sum SFA 28.2 [+ or -] [0.1.sup.d]
Sum MUFA 24.7 [+ or -] [0.1.sup.e]
Sum PUFA 41.5 [+ or -] [0.2.sup.bc]
Sum n-3 25.9 [+ or -] [0.1.sup.acd]
Sum n-6 14.3 [+ or -] [0.0.sup.ab]
Ratio (n-3)/(n-6) 1.8 [+ or -] [0.0.sup.a]
Ratio EPA/AA 1.4 [+ or -] [0.0.sup.cd]
Ratio DHA/EPA 0.8 [+ or -] [0.0.sup.e]

Total FA (mg/g DM) 27.4 [+ or -] [3.2.sup.bc]

 Hatch 2

 Stage II

 Fatty acid Beginning

14:0 1.2 [+ or -] [0.2.sup.ac]
16:1n-7c 3.2 [+ or -] [0.0.sup.cd]
16:0 14.0 [+ or -] [0.2.sup.c]
17:0 0.9 [+ or -] [0.0.sup.bd]
18:2n-6 1.9 [+ or -] [0.0.sup.b]
18:1n-9c 13.8 [+ or -] [0.1.sup.bc]
18:1n-7c 10.4 [+ or -] [0.5.sup.e]
18:0 11.8 [+ or -] [0.1.sup.c]
18:0 Fade 1.5 [+ or -] [0.1.sup.c]
20:4n-6 AA 7.4 [+ or -] [0.3.sup.cd]
20:5n-3 EPA 14.9 [+ or -] [0.1.sup.ac]
20:2n-6 0.5 [+ or -] [0.0.sup.b]
20:1(n-9/11)c 1.0 [+ or -] [0.0.sup.cd]
20:0 0.8 [+ or -] [0.1.sup.bc]
22:5n-6 1.6 [+ or -] [0.1.sup.c]
22:6n-3 DHA 7.9 [+ or -] [0.2.sup.bd]
22:5n-3 0.2 [+ or -] [0.0.sup.ce]
22:0 1.2 [+ or -] [0.1.sup.cd]
Other 6.4 [+ or -] 0.1

Sum SFA 31.6 [+ or -] [0.3.sup.bc]
Sum MUFA 29.9 [+ or -] [0.5.sup.bc]
Sum PUFA 26.0 [+ or -] [0.2.sup.de]
Sum n-3 23.7 [+ or -] [0.2.sup.cd]
Sum n-6 11.9 [+ or -] [0.2.sup.cd]
Ratio (n-3)/(n-6) 2.0 [+ or -] [0.0.sup.ab]
Ratio EPA/AA 2.0 [+ or -] [0.1.sup.b]
Ratio DHA/EPA 0.5 [+ or -] [0.0.sup.cd]

Total FA (mg/g DM) 33.1 [+ or -] [0.9.sup.c]

 Hatch 2

 Stage II

 Fatty acid Fed

14:0 1.4 [+ or -] [0.1.sup.c]
16:1n-7c 3.7 [+ or -] [0.1.sup.de]
16:0 15.2 [+ or -] [0.3.sup.d]
17:0 0.9 [+ or -] [0.0.sup.ab]
18:2n-6 1.9 [+ or -] [0.0.sup.b]
18:1n-9c 14.5 [+ or -] [0.1.sup.b]
18:1n-7c 12.0 [+ or -] [0.1.sup.f]
18:0 11.7 [+ or -] [0.1.sup.c]
18:0 Fade 0.8 [+ or -] [0.0.sup.e]
20:4n-6 AA 5.8 [+ or -] [0.0.sup.d]
20:5n-3 EPA 14.2 [+ or -] [0.2.sup.ac]
20:2n-6 0.3 [+ or -] [0.0.sup.d]
20:1(n-9/11)c 0.8 [+ or -] [0.0.sup.de]
20:0 0.6 [+ or -] [0.0.sup.ad]
22:5n-6 1.9 [+ or -] [0.1.sup.d]
22:6n-3 DHA 7.8 [+ or -] [0.3.sup.bd]
22:5n-3 0.2 [+ or -] [0.0.sup.ce]
22:0 1.0 [+ or -] [0.0.sup.bd]
Other 5.8 [+ or -] 0.1

Sum SFA 32.5 [+ or -] [0.4.sup.bc]
Sum MUFA 32.5 [+ or -] [0.2.sup.c]
Sum PUFA 33.6 [+ or -] [0.7.sup.e]
Sum n-3 22.9 [+ or -] [0.5.sup.cd]
Sum n-6 10.5 [+ or -] [0.1.sup.de]
Ratio (n-3)/(n-6) 2.2 [+ or -] [0.0.sup.bc]
Ratio EPA/AA 2.4 [+ or -] [0.0.sup.e]
Ratio DHA/EPA 0.6 [+ or -] [0.0.sup.d]

Total FA (mg/g DM) 57.8 [+ or -] [7.5.sup.d]

 Hatch 2

 Stage II

 Fatty acid Starved

14:0 1.1 [+ or -] [0.2.sup.ac]
16:1n-7c 1.8 [+ or -] [0.1.sup.bg]
16:0 12.6 [+ or -] [0.1.sup.a]
17:0 1.0 [+ or -] [0.0.sup.bc]
18:2n-6 1.5 [+ or -] [0.1.sup.c]
18:1n-9c 10.6 [+ or -] [0.2.sup.d]
18:1n-7c 8.2 [+ or -] [0.1.sup.g]
18:0 13.9 [+ or -] [0.1.sup.d]
18:0 Fade 2.3 [+ or -] [0.2.sup.ab]
20:4n-6 AA 9.8 [+ or -] [0.2.sup.ab]
20:5n-3 EPA 16.4 [+ or -] [0.1.sup.ac]
20:2n-6 0.7 [+ or -] [0.1.sup.a]
20:1(n-9/11)c 1.0 [+ or -] [0.0.sup.cd]
20:0 1.1 [+ or -] [0.0.sup.f]
22:5n-6 1.2 [+ or -] [0.0.sup.f]
22:6n-3 DHA 8.6 [+ or -] [0.1.sup.be]
22:5n-3 0.1 [+ or -] [0.0.sup.f]
22:0 1.8 [+ or -] [0.0.sup.e]
Other 7.3 [+ or -] 0.7

Sum SFA 33.0 [+ or -] [0.6.sup.b]
Sum MUFA 22.8 [+ or -] [0.3.sup.ae]
Sum PUFA 40.2 [+ or -] [0.2.sup.bcd]
Sum n-3 25.8 [+ or -] [0.2.sup.acd]
Sum n-6 13.6 [+ or -] [0.1.sup.bc]
Ratio (n-3)/(n-6) 1.9 [+ or -] [0.0.sup.ab]
Ratio EPA/AA 1.7 [+ or -] [0.0.sup.abc]
Ratio DHA/EPA 0.5 [+ or -] [0.0.sup.cd]

Total FA (mg/g DM) 14.0 [+ or -] [0.7.sup.a]

 Hatch 2

 Stage III

 Fatty acid Beginning

14:0 1.2 [+ or -] [0.1.sup.ac]
16:1n-7c 3.8 [+ or -] [0.2.sup.e]
16:0 14.6 [+ or -] [0.3.sup.cd]
17:0 1.1 [+ or -] [0.0.sup.cd]
18:2n-6 1.9 [+ or -] [0.0.sup.b]
18:1n-9c 13.6 [+ or -] [0.2.sup.bc]
18:1n-7c 12.3 [+ or -] [0.2.sup.f]
18:0 12.8 [+ or -] [0.2.sup.cd]
18:0 Fade 1.0 [+ or -] [0.0.sup.e]
20:4n-6 AA 6.0 [+ or -] [0.1.sup.d]
20:5n-3 EPA 14.6 [+ or -] [0.2.sup.ac]
20:2n-6 0.3 [+ or -] [0.0.sup.d]
20:1(n-9/11)c 0.7 [+ or -] [0.0.sup.e]
20:0 0.7 [+ or -] [0.0.sup.cd]
22:5n-6 1.6 [+ or -] [0.1.sup.c]
22:6n-3 DHA 7.0 [+ or -] [0.3.sup.d]
22:5n-3 0.1 [+ or -] [0.0.sup.ef]
22:0 1.2 [+ or -] [0.01.sup.d]
Other 6.1 [+ or -] 0.2

Sum SFA 33.2 [+ or -] [0.4.sup.b]
Sum MUFA 31.9 [+ or -] [0.5.sup.c]
Sum PUFA 32.9 [+ or -] [0.8.sup.e]
Sum n-3 22.3 [+ or -] [0.6.sup.d]
Sum n-6 10.2 [+ or -] [0.2.sup.e]
Ratio (n-3)/(n-6) 2.2 [+ or -] [0.0.sup.bc]
Ratio EPA/AA 2.4 [+ or -] [0.0.sup.e]
Ratio DHA/EPA 0.5 [+ or -] [0.0.sup.c]

Total FA (mg/g DM) 33.7 [+ or -] [2.9.sup.c]

Data, within a row, not sharing a common Superscript are significantly
different (P < 0.05).

Other <1%: 12:0, i14:0, 14:1, C14PUFA, i15:0, a15:0, 15:1, 15:0, i16:0,
C16PUFA, 16:1n-9c, 16:2, 16:1n-5c, 16:0 Fade, br17:1, i17:0, a17:0,
17:1, 18:3n-6, i18:0, 18:4n-3, 18:3n-3, 18:1n-5c, C19PUFA, i19:0, 19:1,
20:2, 20:3n-6, 20:2NMI, 20:4n-3 20:1n-7c, C20Falde, C21PUFA, 21:0,
22:4n-6, 22:1, C28PUFA.

AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic
acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids;
PUFA, polyunsaturated fatty acids; NMI, non-methylene interrupted;
Fade, fatty aldehyde; DM, dry mass.


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GRANT C. LIDDY, (1) MATTHEW M. NELSON, (2) PETER D. NICHOLS, (3) BRUCE F. PHILLIPS (1) AND GREG B. MAGUIRE (4)

(1) Aquatic Science Research Unit, Curtin University of Technology Perth, W.A. 6845, Australia; (2) Department of Zoology University of Tasmania, Hobart, Tasmania, 7001, Australia; (3) CSIRO Marine Research, Hobart, Tasmania, 7001, Australia: (4) Department of Fisheries, Research Division, North Beach, W.A. 6920, Australia
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Date:Apr 1, 2004
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