Fatty acid composition and utilization in developing eggs of some marine nudibranchs (mollusca: gastropoda: opistobranchia) from southwest Spain.

ABSTRACT The fatty acid composition of whole egg masses was investigated in 4 marine nudibranchs collected from the southwest of Spain: Polycera aurantiomarginata, Polycera quadrilineata, Berghia columbina and Berghia verrucicornis. The four species are carnivorous. All nudibranchs were characterized by high levels of n-3 polyunsaturated fatty acids (n-3 PUFA), mainly eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3) and docosahexaenoic acid (22:6n-3). Other major fatty acids were the saturated palmitic (16:0) and stearic (18:0) acids, the monounsaturated oleic acid (18:1n-9) and the n-6 PUFA arachidonic (20:4n- 6) and docosatetraenoic (22:4n-6) acids. Relative high percentages of the plasmalogen derivatives 16:0DMA and 18:0DMA were also detected. Univariate analysis showed that egg fatty acids from P. aurantiomarginata and P. quadrilineata significantly differed from those of B. columbina and B. verrucicornis. Higher levels of saturated fatty acids and 22:6n-3 and lower percentages of 18:0DMA, 22:4n-6 and 22:5n-3 were found in the spawns of both species of Polycera as compared with both of Berghia. Analysis of the bryozoon Bugula neritina and the sea anemone Sagartia troglodites, main preys of Polycera and Berghia respectively, showed that the differences observed in egg fatty acid composition were likely related to diet. Fatty acids of the fully developed embryos from P. aurantiomarginata and B. columbina, sampled immediately prior to hatching, were analyzed to investigate the dynamics of fatty acids during embryogenesis. The long chain PUFA 20:4n-6, 22:4n-6, 20:5n-3 and 22:5n-3 increased or remained stable during embryogenesis in both species whereas palmitoleic acid (16:1n-7) in B. columbina and 16:0, 17:0, 18:0 and 18:1n-9 in P. aurantiomarginata decreased. The major n-3 PUFA, 22:6n-3, marked decreased in B. columbina but remained unchanged in P. aurantiomarginata. These data indicate that fatty acids play different roles during embryogenesis and that in both nudibranchs the embryo requirements are not the same.

KEY WORDS: eggs, embryogenesis, fatty acids, gastropoda, nudibranchia, opistobranchia

INTRODUCTION

In molluscs, lipids laid down in the eggs play an important role during embryogenesis and early larval development because they are used as a source of energy and as structural components for membrane formation. Although it has been shown in bivalves that hatching success and larval survival is related to egg lipid content (Utting & Millican 1997), lipid quality in terms of fatty acid composition must be also considered. Diets supplemented with 20:5n-3 (EPA) and/or 22:6n-3 (DHA) have been proved to enhance fecundity, hatching rates and larval survival in different species of bivalves (Soudant et al. 1996a, Utting & Millican 1997, Caers et al. 1999a, Caers et al. 2002, Hendriks et al. 2003, Nevejan et al. 2003) and in the gastropod Haliotis asinina (Bautista-Teruel et al. 2001). Both fatty acids, which are usually the major polyunsaturated fatty acids (PUFA) found in marine invertebrates, are important components of membranes where they contribute to its integrity. Additionally, EPA can be a source of energy because it has been reported to decrease during embryonic and larval development (Whyte et al. 1990, Whyte et al. 1991, Marty et al. 1992, Soudant et al. 1998, Labarta et al. 1999). This fatty acid, together with arachidonic acid (20:4n-6), is also a precursor of prostaglandins, a group of biologically active compounds that participates in marine invertebrate reproduction (Osada et al. 1989, Di Matzo et al. 1992, Martinez et al. 1999, Martinez et al. 2000).

Tissue fatty acid composition can be considered as the result of endogenous synthesis and exogenous supply. The essential fatty acids linoleic (18:2n-6) and alpha-linolenic (18:3n-3) cannot be synthesized in animal tissues and must be provided by diet. With respect to other PUFA, previous studies have shown that bivalves (Waldock & Holland 1984, Laing et al. 1990, Chu & Greaves 1991, Delaunay et al. 1993, Nevejan et al. 2003) and the gastropod Haliotis (Uki et al. 1986, Dunstan et al. 1993, Durazo-Beltran et al. 2003) are able to elongate and desaturate 18:2n-6 and 18:3n-3 to obtain 20:4n-6, EPA and DHA; but this capacity is usually too low to fulfill the species' requirements, and these PUFA have also to be obtained through the diet. Therefore, it is not surprising that dietary lipids strongly influence the fatty acid composition of these marine invertebrates and, in turn, that of their spawned eggs. In bivalves (Soudant et al. 1996a, Soudant et al. 1996b, Caers et al. 1999a, Caers et al. 2002, Caers et al. 2003, Duinker et al. 2004, Ojea et al. 2004) and in Haliotis (Bautista-Teruel et al. 2001, Nelson et al. 2002, Brazao et al. 2003), it has been found that fatty acid composition of gonads and eggs is related to that of diet.

The fatty acid composition of mollusc eggs and larvae has been mainly studied in bivalves, especially in those species commonly reared in commercial hatcheries such as oysters, scallops or clams. In gastropods, fatty acids have been characterized in adult tissues but they have not been investigated in the eggs and in larvae, there is only one study in the abalone Haliotis fulgens (Nelson et al. 2002). Benkendorff et al. (2005) in a recent study, investigated the fatty acids of egg masses in 16 species of gastropods. This study was focused on their antimicrobial properties, and only free fatty acids were analyzed.

In this study, fatty acid composition of the gelatinous egg masses of four species of nudibranchs, Polycera aurantiomarginata, Polycera quadrilineata, Berghia columbina and Berghia verrucicornis, has been characterized. To investigate the utilization of fatty acids during embryogenesis in the species P. aurantiomarginata and B. columbina, analysis was performed on freshly-laid spawns, with uncleaved fertilized eggs, and on spawns with fully developed embryos sampled immediately prior to hatching. To know whether dietary lipids influenced egg fatty acid composition, the bryozoon Bugula neritina and the sea anemone Sagartia troglodites, main prey of both species of Polycera and both species of Berghia respectively, were also analyzed.

MATERIALS AND METHODS

Sample Collection

Adults of four species of nudibranchs: Polycera aurantiomarginata, Polycera quadrilineata, Berghia columbina and Berghia verrucicornis were collected by hand from the same intertidal area of El Portil, located in the estuary area of the river Piedras (Huelva, SW Spain) (37[degrees]12'40"N, 7[degrees]7'50"W) (Fig. 1). Polycera aurantiomarginata, an abundant species in the studied area where it reproduces all along the year, was collected from December 2001 to December 2002. Polycera quadrilineata is a very scarce species that was only found from May to June 2002. Berghia columbina and B. verrucicornis are present in the area almost all the year, but they have a short reproductive period that spreads from March to June; specimens of both species were collected during this period in 2002 and 2003. The individuals were carried to the laboratory where they were measured and placed in pairs in beakers with 500 mL of seawater. The adults of each pair showed similar size. They were maintained at a constant temperature (19[degrees]C) and photoperiod (16 h light and 8 of dark (16L:SD)).

[FIGURE 1 OMITTED]

The egg masses with uncleaved eggs laid by the adults of the four species of nudibranchs were collected and immediately frozen at -80[degrees]C until analysis. Sixty-two samples were collected from P. aurantiomarginata and nine from the other three species. In the embryogenesis study, spawns from P. aurantiomarginata and B. columbina were isolated in beakers containing 600 mL of filtered seawater and maintained in the same conditions of temperature and photoperiod as the adults. The water was renewed every day. When the veligers were developed the spawns were frozen and stored at -80[degrees]C until fatty acid analysis. Nineteen samples were analyzed in P. aurantiomarginata and nine in B. columbina.

Fatty Acid Analysis

Individual whole spawns with uncleaved eggs and veligers and specimens of Bugula neritina and Sagartia troglodites were homogenized in 0.2 mL of 0.9% NaCl. Fatty acid methyl esters were prepared by a direct transesterification reaction according to Lepage and Roy (1987). Briefly, 2 mL of methanol-benzene 4:1 (v/v) was added to 0.2 mL of homogenate and then, while stirring, 0.2 mL of acetyl chloride was slowly added. Tubes were tightly closed and maintained at 100[degrees]C for 1 h. After cooling, 5 mL of 6% [K.sub.2]C[O.sub.3] solution was added. The tubes were then shaken and centrifuged and the benzene upper phase was recovered for fatty acid analysis.

Fatty acid methyl esters were separated by gas-liquid chromatography using a Hewlett Packard 5890 gas chromatograph equipped with a fused silica capillary column (30 m x 0.25 mm ID) coated with TR-WAX and supplied by Teknokroma (Barcelona, Spain). Peaks were identified by comparison with known standards (FAME mix C4-C24, Supelco/Sigma-Aldrich), and with a well characterized profile of menhaden fish oil (Supelco/Sigma-Aldrich); the results were reported as area percentages.

Statistical Analysis

Values were expressed as means [+ or -] SD. One-way ANOVA followed by the Student-Newman-Keuls test were used to evaluate the significant differences in egg fatty acid composition between species. Additionally, unpaired Student t-test was used to determine differences between mean values for fatty acids during embryogenesis and between Bugula neritina and Sagartia troglodites.

RESULTS

The fatty acid composition of Bugula neritina and Sagartia troglodites is shown in Table 1. The sea anemone showed higher percentages of polyunsaturated fatty acids (PUFA) and lower of saturated fatty acids (SAFA) than B. neritina. Both invertebrates had high levels of EPA and DHA although in B. neritina the amount of DHA was twice as high as EPA whereas in S. troglodites EPA clearly predominated over DHA. The PUFA profile of the sea anemone was also characterized by the elevated levels of 22:5n-3 (5.7%) and 22:4n-6 (10.6%), two fatty acids that in B. neritina accounted for only 1%. In both species palmitic (16:0) and stearic (18:0) were the major saturated fatty acids although in B. neritina 14:0 was also elevated. Total monounsaturated fatty acid (MUFA) levels were similar in B. neritina and S. troglodites but significant differences could be observed among specific fatty acids. Palmitoleic (16:1n-7), oleic (18:1n-9) and 20:1n-9 were higher in B. neritina than in S. troglodites but the sea anemone showed two additional fatty acids, 20:1n-7 and 22:1n-9, which were not detected in B. neritina. Two dimethyl acetals, 16:0DMA and 18:0DMA, were also found in both marine invertebrates and both were significantly higher in S. troglodites than in B. neritina.

Egg masses from Polycera aurantiomarginata, Polycera quadrilineata, Berghia columbina and Berghia verrucicornis showed a fatty acid profile (Table 2) characterized by a high content of PUFA (44-50%), mainly n-3 derivatives (31-34%), followed by SAFA (25-35%). Total MUFA were lower, accounting for less than 15%. Significant differences were found among the four studied species although these differences were more marked between Polycera and Berghia than between the two species of each genus. Total n-3 PUFA showed similar values in the eggs of the four nudibranchs and were dominated by EPA and DHA. Berghia columbina and B. verrucicornis had lower values of DHA than P. aurantiomarginata and P. quadrilineata whereas EPA levels were more variable and ranged from 8.7% in P. aurantiomarginata to 12.6% in B. verrucicornis. However the most striking differences were found for 22:5n-3 whose percentages were three times greater in both species of Berghia than in both of Polycera. Total n-6 PUFA showed lower values in Polycera than in Berghia. Arachidonic acid, which is usually the main component of this family of fatty acids, showed similar proportions in the four species (3% to 4%) but marked differences was observed for 22:4n-6 accounting for 7-9% in both species of Berghia and only for 2% in Polycera. Low percentages of two n-4 PUFA, 16:2n-4 and 16:3n-4 were also found in the eggs of the four studied nudibranchs.

The major saturated fatty acids found in the eggs of the four species of nudibranchs were 16:0, 17:0 and 18:0. The level of 17:0 and 18:0 was marked increased in Polycera as compared with Berghia but these two fatty acids were also higher in P. aurantiomarginata than in P. quadrilineata. High percentages of 18:0DMA were observed in all species and were higher in Berghia than in Polycera. Monounsaturated fatty acids were represented by 16:1n-7 and C-18 and C20 components of n-7 and n-9 series. Oleic acid was the main MUFA and showed similar values in the four studied species. However, significant differences were found for 18:1n-7, which was higher in Polycera than in Berghia and for 20:1n-7 whose level was much lower in Polycera. In addition, eggs from B. columbina showed the highest percentages of 16:1n-7 and 20:1n-7.

The fatty acid profile of fully developed embryos from P. aurantiomarginata and B. columbina, analyzed immediately prior to hatching, is shown in Table 3. When values of veliger fatty acids were compared with those found in eggs (Fig. 2) it could be observed that the behavior of these two species of nudibranchs clearly differed from each other. Total n-3 PUFA significantly decreased during embryogenesis in B. columbina, mainly because of the reduction in DHA, whereas the level of these fatty acids remained stable in P. aurantiomarginata. In both species EPA was not significantly modified whereas its long-chain derivative, 22: 5n-3, increased. Higher levels of total n-6 PUFA were found in the embryos of P. aurantiomarginata and B. columbina as compared with eggs. In the former, this increase was mainly because of 22:4n-6. However in B. columbina, the three major fatty acids of this family of PUFA, 20:4n-6, 22:4n-6 and 22:5n-6, were significantly increased. Finally to point out that the minor n-6 PUFA, linoleic acid, significantly decreased in both species of nudibranchs during embryogenesis.

[FIGURE 2 OMITTED]

Saturated fatty acids showed the most marked changes in P. aurantiomarginata because the three major components, 16:0, 17:0 and 18:0, were significantly decreased in the veligers as compared with eggs. In B. columbina only a significant increase in 18:0 could be observed. Changes observed in MUFA of P. aurantiomarginata during embryonic development were characterized by a marked decrease of 18:1n-9 and an increase of 18:1n-7, 20:1n-7 and 20:1n-9. However, in the embryos of B. columbina the most important change was the marked decrease of 16:1n-7 and the increase of 20:1n-9. In both species of nudibranchs the level of 18:0DMA was significantly increased at the end of embryogenesis.

DISCUSSION

Studies investigating the fatty acid composition of molluscan eggs and larvae have been focused on bivalves because of the high commercial value of some of their species. However, at present, these compounds have not been characterized in marine gastropods, either in freshly-laid spawns or during embryo development. Thus, the first aim of this study is to characterize the fatty acid profile of the gelatinous egg masses of 4 different species of nudibranchs: Polycera aurantiomarginata, Polycera quadrilineata, Berghia columbina and Berghia verrucicornis.

The spawns of all the species examined were characterized by elevated levels of C20 and C22 highly unsaturated fatty acids. This feature, common in marine animals, is considered an adaptation to the relative low temperatures that characterize the marine environment because these fatty acids aid to maintain lipid fluidity. As in other marine invertebrates, high levels of n-3 PUFA were found in the eggs of the studied nudibranchs, with EPA and DHA as major components. High levels of EPA are common in the lipids of gastropods but DHA, which is a major fatty acid in bivalves, is usually low in gastropods (Joseph 1982, Dunstan et al. 1996, Kharlamenko et al. 2001, Nelson et al. 2002, Brazao et al. 2003, Morais et al. 2003, Grubert et al. 2004). Only in the prosobranch Lunatia triseriata (Joseph 1982) and in the polar pelagic opisthobranchs Clione limacina, Limacina helicina and Spongiobranchaea australis (Phleger et al. 1997, Phleger et al. 1999, Kattner et al. 1998) the percentage of DHA was similar to that found in the eggs of Polycera and Berghia. A relative high level of 22:5n-3 (2% to 6%) was also found in the eggs of these nudibranchs. This fatty acid is usually present in gastropods but its percentage greatly varies among species, ranging from about 1% in Patella (Joseph 1982, Brazao et al. 2003, Morais et al. 2003) to more than 9% in Haliotis (Joseph 1982, Dunstan et al. 1996, Nelson et al. 2002, Grubert et al. 2004).

Total n-3 PUFA showed similar values in the eggs of the studied nudibranchs but marked differences could be observed for specific fatty acids. DHA, the major n-3 PUFA, and other minor components such as 18:3n-3, 20:3n-3 and 20:4n-3, were higher in both species of Polycera than in Berghia, whereas the reverse was true for 22:5n-3. The percentage of EPA significantly differed among the four species: P. quadrilineata and B. columbina showed the same value but it was increased in B. verrucicornis and showed the lowest value in P. aurantiomarginata. It has been previously reported in bivalves that dietary lipids greatly influence the fatty acid composition of eggs (Soudant et al. 1996b, Caers et al. 1999a, Caers et al. 2002). Therefore, differences observed in n-3 PUFA of egg masses may be at least partially attributed to the different feeding habits of Polycera and Berghia. Polycera feeds mainly on the bryozoon Bugula neritina, which showed an n-3 PUFA pattern typical of a filter-feeding animal characterized by high levels of DHA, followed by EPA and by minor percentages of 18:3n-3 and other n-3 PUFA. The sea anemone Sagartia troglodites, main prey of B. columbina and B. verrucicornis, had also elevated levels of EPA and DHA, but EPA predominated, and its percentage of 18:3n-3 was significantly lower than in B. neritina. However, the most striking difference between these two invertebrates, which could be also observed between Polycera and Berghia, was the high proportion of 22:5n-3 found in S. troglodites, which was more than five times greater than in B. neritina. Sea anemones are carnivorous but they can also feed on plankton, which could explain the presence of high amounts of EPA and DHA. The increased 22:5n-3 seems to be a specific characteristic of sea anemones as it has been described for other species (Joseph 1979, Pollero 1983).

In addition to a direct dietary influence, it is likely that parents could also control the amount of n-3 PUFA that must be incorporated to eggs to assure its viability, especially that of EPA and DHA, which are considered essential for embryo development. It has been reported in bivalves that eggs from animals fed an EPA deficient diet showed similar EPA levels to those from animals fed a high EPA diet (Caers et al. 1999a, Caers et al. 2002) indicating a specific accumulation of this fatty acid in the eggs irrespective of its presence in the diet. On the other hand, the amount of DHA that can be transferred to the eggs seems to be limited because Caers et al. (1999a, 2002) observed in Argopecten purpuratus and Crassostrea gigas that the eggs obtained from adults fed high levels of DHA (45%) did not show elevated levels of this fatty acid. Data obtained in the present study also indicate that nudibranchs may control EPA and DHA levels in eggs because, for example, the percentage of DHA in B. columbina and B. verrucicornis exceeded that found in S. troglodites.

Arachidonic acid and 22:4n-6 were the major n-6 PUFA in the spawns of the four species of nudibranchs. Arachidonic acid is common in macroalgae (Li et al. 2002) and thus is found in substantial amounts in herbivorous gastropods (Kharlamenko et al. 2001, Nelson et al. 2002, Brazao et al. 2003, Morais et al. 2003, Grubert et al. 2004). In the present study, the level of 20:4n-6 was below that found in these prosobranchs but was higher than that reported for polar opisthobranchs (Phleger et al. 1997, Phleger et al. 1999, Kattner et al. 1998, Falk-Petersen et al. 2001). Although the amount of 20:4n-6 in S. troglodites almost doubled that of B. neritina, this fatty acid showed similar levels in the eggs of the four studied nudibranchs. This enrichment of arachidonic acid in the eggs of both species of Polycera suggests that this fatty acid may be important for nudibranch embryo development. The second major n-6 PUFA, 22:4n-6, usually found in other molluscs in small amounts, showed a percentage at least three times greater in the eggs of B. columbina and B. verrucicornis than in P. aurantiomarginata and P. quadrilineata. These differences were clearly related to diet because in S. troglodites 22:4n-6 accounted for 10% and only for 1% in B. neritina.

The percentage of total saturated fatty acids ranged from 25% to 35%, with the highest values observed in the eggs of P. aurantiomarginata. As found in most mollusc species, the main saturated fatty acids in the egg masses of the four nudibranchs were palmitic and stearic, two fatty acids that are normally endogenously synthesized. Because no marked differences were found for 16:0 and 18:0 in B. neritina and S. troglodites, the different levels of both fatty acids observed in the four nudibranchs may depend on the metabolic peculiarities of each species. However, it should be pointed out that the strikingly high percentages of 17:0 found in both species of Polycera (5% to 6%), an odd chain fatty acid that in other gastropod species, does not usually exceed 1%. The exception is the polar opisthobranch Clione limacine, which has been reported to present a percentage of 17:0 up to 4% (Phleger et al. 1997, Phleger et al. 2001, Kattner et al. 1998). Two major mechanisms could be proposed to explain the origin of this fatty acid: dietary uptake and "de novo" biosynthesis. The first mechanism is unlikely because the percentage of 17:0 in B. neritina, although higher than that of S. troglodites, was low. Hence, the possibility that the adults of P. aurantiomarginata and P. quadrilineata were able to synthesize this fatty acid must be considered. The synthesis of 17:0 and other odd chain fatty acids needs propionate as initial molecule. In Clione limacina, Kattner et al. (1998) suggested that this acid may originate from the planktonic dimethyl-[beta]-propiothetin (DMPT), a compound which was shown to accumulate in its prey Limacina helicina (Levasseur et al. 1994). The bryozoon B. neritina, as L. helicina, feeds on plankton and therefore the presence of DMPT, although not investigated, cannot be discarded.

Palmitoleic, oleic and vaccenic acids are usually the major monounsaturated fatty acids found in gastropods, although their concentrations greatly vary among the different species (Joseph 1982, Phleger et al. 1997, Phleger et al. 2001, Kattner et al. 1998, Kharlamenko et al. 2001, Nelson et al. 2002, Brazao et al. 2003). In the eggs of the four studied nudibranchs, these fatty acids were also dominant. Egg masses from B. columbina showed the highest percentages of 16:1n-7. The level of this fatty acid in S. troglodites was less than 2%, even lower than that of B. neritina. Therefore, palmitoleic was probably synthesized by parents and then selectively accumulated in the eggs. Oleic acid, the major monounsaturated fatty acid, showed a percentage that was at least twice as high as vaccenic acid. This is consistent with the carnivorous feeding habits of the studied nudibranchs because vaccenic acid is found in marine bacteria (Brown et al. 1996) and some species of macroalgae (Li et al. 2002) and thus its level is usually higher in herbivorous gastropods (Nelson et al. 2002, Brazao et al. 2003, Morais et al. 2003, Grubert et al. 2004).

Two dimethyl acetals, 16:0DMA and 18:0DMA, were found in the spawns of the studied nudibranchs. These compounds originate from transmethylation of 1-alkenyl chains of plasmalogens, a type of ether-containing phosphoglycerides found in animal membrane cells. The abundance of plasmalogens in molluscs has been previously reported (Joseph 1982, Chapelle 1987, Kraffe et al. 2004) but information concerning their biologic function is still limited. They are supposed to be important in membrane dynamics (Nagan & Zoeller 2001), and in mammalian cells they may also serve as antioxidants (Brosche & Platt 1998). In the egg masses 18:0DMA was the major dimethyl acetal accounting for up to 9% whereas 16:0DMA percentages were less than 3%. To our knowledge there is no information about the alkenyl chain composition in marine gastropods although in two freshwater prosobranchs, Misra et al. (2002) found 16:0DMA and 18:0DMA as the main alkenyl derivatives. Our results are also in agreement with those reported in the eggs of two species of bivalves (Caers et al. 1999a, Caers et al. 2002) in which the quantitatively most important alkenyl chain was 18:0. The levels of 18:0DMA were higher in both species of Berghia than in Polycera and equaled those found in their respective preys, S. troglodites and B. neritina.

Previous studies performed on bivalves have proved the importance of fatty acids during embryo and larval development (Whyte et al. 1990, Whyte et al. 1991, Delaunay et al. 1993, Utting & Millican 1997, Labarta et al. 1999, Pernet & Tremblay 2004), either as a source of energy or as structural components. In bivalves, EPA is considered to have an energy-providing role because it has been shown to decrease during embryo and larval development (Whyte et al. 1990, Whyte et al. 1991, Soudant et al. 1998, Labarta et al. 1999). However, in the veligers of P. aurantiomarginata and B. columbina, the level of this fatty acid did not significantly differ from that found in the uncleaved eggs. Nevertheless there were some fatty acids that decreased during embryogenesis: 16:0, 17:0, 18:0 and 18:1n-9 in P. aurantiomarginata and 16:1n-7 and DHA in B. columbina The decline of 18:1n-9 in P. aurantiomarginata could caused by, at least in part, its elongation to 20:1n-9 because the level of this fatty acid in embryos doubled that of eggs. The decrease of DHA has not been reported in other molluscs; in bivalves, this fatty acid has a major structural role and, as observed in this study for P. aurantiomarginata, its level remains constant during embryogenesis (Whyte et al. 1990, Whyte et al. 1991, Marty et al. 1992, Labarta et al. 1999, Leonardos & Lucas 2000).

The long-chain PUFA 22:4n-6 and 22:5n-3 has been reported to increase during embryogenesis (Whyte et al. 1990, Whyte et al. 1991, Labarta et al. 1999) and to be retained by larvae during starvation (Delaunay et al. 1993, Caers et al. 1998, Caers et al. 1999b). In P. aurantiomarginata and B. columbina both fatty acids may be important for embryos because, as stated later, they are present in the spawns in high percentages and during embryogenesis they significantly increased.

In summary, results from this study indicate that in nudibranchs, as in other molluscs, the feeding habits of the parents markedly influence the fatty acid composition of the spawns, al though some long-chain PUFA such as 20:4n-6, 20:5n-3 and 22:6n-3, considered essentials for embryo development, could be selectively accumulated in the eggs to assure their viability. On the other hand, the differences observed in fatty acid composition between uncleaved eggs and veligers in P. aurantiomarginata and B. columbina indicate that these compounds are important for embryo development, either as energy source or as structural components.

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INES MARTINEZ-PITA, (1) FRANCISCO GARCIA (1) AND MARIA-LUISA PITA (2)*

(1) Departamento de Fisiologia y Zoologia, Universidad de Sevilla, Avda. Reina Mercedes, 6, 41012 Sevilla, Spain; (2) Departamento de Bioquimica Medica y Biologia Molecular, Universidad de Sevilla, Avda, Sanchez Pizjuan, 4, 41009 Sevilla, Spain

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Fatty acid composition (as % of total fatty acids) of Bugula neritina
and Sagartia troglodites.

                              Bugula               Sagartia
                             neritina             troglodites
Fatty acids                   (n = 4)               (n = 4)

14:0                     3.1 [+ or -] 0.1    1.0 [+ or -] 0.1 (a)
15:0                     0.5 [+ or -] 0.0    0.6 [+ or -] 0.0 (a)
16:0                     21.1 [+ or -] 0.0   17.3 [+ or -] 0.9 (a)
17:0                     2.3 [+ or -] 0.0    1.7 [+ or -] 0.1 (a)
18:0                     8.9 [+ or -] 0.1    7.5 [+ or -] 0.4 (a)
20:0                     1.9 [+ or -] 0.0             --
22:0                     0.9 [+ or -] 0.1    0.4 [+ or -] 0.1 (a)
[SIGMA]saturates         38.7 [+ or -] 0.4   26.8 [+ or -] 1.2 (a)
16:1n-7                  2.9 [+ or -] 0.0    1.6 [+ or -] 0.4 (a)
18:1n-7                  2.1 [+ or -] 0.0      1.7 [+ or -] 0.4
18:1n-9                  3.9 [+ or -] 0.1    2.0 [+ or -] 0.2 (a)
20:1n-7                         --             2.4 [+ or -] 0.2
20:1n-9                  0.8 [+ or -] 0.0    0.5 [+ or -] 0.1 (a)
22:1n-9                         --             0.8 [+ or -] 0.1
[SIGMA]monounsaturates   9.8 [+ or -] 0.1      9.4 [+ or -] 0.9
18:3n-3                  2.2 [+ or -] 0.1    0.8 [+ or -] 0.3 (a)
18:4n-3                  1.7 [+ or -] 0.1    1.0 [+ or -] 0.2 (a)
20:3n-3                  0.4 [+ or -] 0.1             --
20:4n-3                  1.0 [+ or -] 0.0    0.5 [+ or -] 0.2 (a)
20:5n-3                  7.9 [+ or -] 0.1    14.3 [+ or -] 1.l (a)
22:5n-3                  1.0 [+ or -] 0.1    5.7 [+ or -] 0.5 (a)
22:6n-3                  18.2 [+ or -] 0.5   9.3 [+ or -] 1.0 (a)
[SIGMA]PUFAn-3           30.7 [+ or -] 0.6     31.7 [+ or -] 2.0
16:2n-4                  1.1 [+ or -] 0.0      1.4 [+ or -] 0.3
16:3n-4                  0.4 [+ or -] 0.1             --
[SIGMA]PUFAn-4           1.5 [+ or -] 0.1      1.5 [+ or -] 0.1
18:2n-6                  1.6 [+ or -] 0.1    0.9 [+ or -] 0.1 (a)
18:3n-6                  0.3 [+ or -] 0.0    0.4 [+ or -] 0.0 (a)
20:2n-6                  2.0 [+ or -] 0.0    0.8 [+ or -] 0.1 (a)
20:3n-6                  0.1 [+ or -] 0.0             --
20:4n-6                  1.7 [+ or -] 0.0    3.4 [+ or -] 0.3 (a)
22:4n-6                  1.0 [+ or -] 0.0    10.6 [+ or -] 1.5 (a)
22:5n-6                  1.5 [+ or -] 0.0    0.4 [+ or -] 0.0 (a)
[SIGMA]PUFAn-6           8.2 [+ or -] 0.1    16.5 [+ or -] 1.5 (a)
[SIGMA]polyunsaturates   40.4 [+ or -] 0.6   49.6 [+ or -] 1.0 (a)
16:0DMA                  2.5 [+ or -] 0.1    4.2 [+ or -] 0.5 (a)
18:0DMA                  6.8 [+ or -] 0.1    8.7 [+ or -] 0.7 (a)

Values are mean [+ or -] S.D. n = number of samples.
a: P < 0.05 with respect to B. neritina.

TABLE 2.
Fatty acid composition (as % of total fatty acids) of egg masses
from P. aurantiomarginata, P. quadrilineata, B. columbina and
B. verrucicornis.

                               P. aurantio            P. quadrilineata
                                marginata                 (n = 9)
Fatty acids                     (n = 62)

14:0                      0.7 [+ or -] 0.2 (a)    1.0 [+ or -] 0.26 (b)
15:0                      1.4 [+ or -] 0.2 (a)    1.7 [+ or -] 0.36 (b)
16:0                     15.6 [+ or -] 2.0 (a)   15.7 [+ or -] 1.4 (ac)
17:0                      6.4 [+ or -] 1.0 (a)    5.7 [+ or -] 0.76 (b)
18:0                     11.1 [+ or -] 1.0 (a)    8.8 [+ or -] 1.0 (b)
[SIGMA]saturates         35.3 [+ or -] 2.9 (a)   32.9 [+ or -] 1.3 (b)
16:1n-7                   2.4 [+ or -] 1.1 (a)    2.0 [+ or -] 0.7 (a)
18:1n-7                   2.2 [+ or -] 0.5 (a)    2.2 [+ or -] 0.5 (a)
18:1n-9                   5.9 [+ or -] 1.8 (a)    6.8 [+ or -] 1.2 (a)
20:1n-7                   0.5 [+ or -] 0.2 (a)    0.6 [+ or -] 0.2 (a)
20:1n-9                   0.7 [+ or -] 0.3 (a)    1.4 [+ or -] 0.5 (b)
22:1n-9                            --                        --
[SIGMA]monounsaturates   11.9 [+ or -] 2.3 (a)   13.0 [+ or -] 1.6 (b)
18:3n-3                   0.7 [+ or -] 0.3 (a)    1.2 [+ or -] 0.6 (b)
20:3n-3                   0.5 [+ or -] 0.2 (a)    0.7 [+ or -] 0.2 (b)
20:4n-3                   0.7 [+ or -] 0.3 (a)    0.8 [+ or -] 0.5 (a)
20:5n-3                   8.7 [+ or -] 1.5 (a)   10.5 [+ or -] 0.8 (b)
22:5n-3                   2.0 [+ or -] 0.5 (a)    2.1 [+ or -] 0.6 (b)
22:6n-3                  19.3 [+ or -] 2.5 (a)   18.2 [+ or -] 2.3 (a)
[SIGMA]PUFAn-3           31.9 [+ or -] 4.3       33.6 [+ or -] 3.8
16:2n-4                   1.6 [+ or -] 0.7 (a)    0.6 [+ or -] 0.1 (b)
16:3n-4                   0.8 [+ or -] 0.3 (a)    0.9 [+ or -] 0.3 (a)
[SIGMA]PUFAn-4            2.4 [+ or -] 0.7 (a)    1.5 [+ or -] 0.3 (b)
18:2n-6                   0.9 [+ or -] 0.2 (a)    1.1 [+ or -] 0.2 (b)
18:3n-6                   0.2 [+ or -] 0.1 (a)    0.2 [+ or -] 0.1
20:2n-6                   1.4 [+ or -] 0.2 (a)    1.2 [+ or -] 0.3 (b)
20:3n-6                   0.2 [+ or -] 0.1        0.2 [+ or -] 0.1
20:4n-6                   3.5 [+ or -] 1.1        4.4 [+ or -] 1.8
22:4n-6                   2.4 [+ or -] 0.8 (a)    2.7 [+ or -] 1.1 (a)
22:5n-6                   1.7 [+ or -] 0.7 (a)    1.2 [+ or -] 0.4 (b)
[SIGMA]PUFAn-6           10.3 [+ or -] 1.7 (a)   11.1 [+ or -] 2.6 (a)
[SIGMA]polyunsaturates   44.7 [+ or -] 3.6 (a)   46.2 [+ or -] 1.2 (a)
16:0DMA                   2.6 [+ or -] 0.4 (a)    2.5 [+ or -] 0.3 (a)
18:0DMA                   5.5 [+ or -] 0.8 (a)    5.5 [+ or -] 0.7 (a)

                               B. columbina            B. verrucicornis

Fatty acids                      (n = 9)                  (n = 9)

14:0                     0.8 [+ or -] 0.1 (a)     0.6 [+ or -] 0.1 (a)
15:0                     1.0 [+ or -] 0.1 (c)     0.5 [+ or -] 0.0 (d)
16:0                    17.5 [+ or -] 0.86 (bc)  17.3 [+ or -] 0.8 (bc)
17:0                     2.2 [+ or -] 0.2 (c)     2.4 [+ or -] 0.1 (c)
18:0                     4.4 [+ or -] 0.3 (c)     6.4 [+ or -] 0.4 (d)
[SIGMA]saturates        25.9 [+ or -] 1.0 (c)    27.3 [+ or -] 1.3 (c)
16:1n-7                  4.3 [+ or -] 1.4 (b)     2.9 [+ or -] 0.4 (a)
18:1n-7                  1.3 [+ or -] 0.2 (b)     1.7 [+ or -] 0.2 (b)
18:1n-9                  5.1 [+ or -] 0.8 (ac)    4.0 [+ or -] 0.3 (bc)
20:1n-7                  3.0 [+ or -] 0.4 (b)     2.1 [+ or -] 0.2 (c)
20:1n-9                  0.5 [+ or -] 0.0 (a)     0.7 [+ or -] 0.1 (a)
22:1n-9                  0.5 [+ or -] 0.1         0.4 [+ or -] 0.1
[SIGMA]monounsaturates  14.8 [+ or -] 2.3 (b)    11.7 [+ or -] 0.7 (a)
18:3n-3                  0.3 [+ or -] 0.1 (c)     0.2 [+ or -] 0.1 (c)
20:3n-3                  0.3 [+ or -] 0.1 (c)     0.4 [+ or -] 0.1 (c)
20:4n-3                  0.3 [+ or -] 0.1 (c)     0.4 [+ or -] 0.1 (c)
20:5n-3                 10.2 [+ or -] 1.0 (b)    12.6 [+ or -] 0.6 (b)
22:5n-3                  6.2 [+ or -] 0.9 (b)     6.5 [+ or -] 0.6 (b)
22:6n-3                 15.1 [+ or -] 1.6 (b)    14.4 [+ or -] 1.0 (b)
[SIGMA]PUFAn-3          32.4 [+ or -] 1.8        34.5 [+ or -] 2.0
16:2n-4                  1.9 [+ or -] 0.3 (a)     2.0 [+ or -] 0.1 (a)
16:3n-4                  0.4 [+ or -] 0.16 (b)    0.3 [+ or -] 0.1 (b)
[SIGMA]PUFAn-4           2.3 [+ or -] 0.3 (ac)    2.3 [+ or -] 0.1 (ac)
18:2n-6                  0.7 [+ or -] 0.1 (c)     0.5 [+ or -] 0.1 (c)
18:3n-6                  0.3 [+ or -] 0.1         0.4 [+ or -] 0.1
20:2n-6                  0.5 [+ or -] 0.1 (c)     0.6 [+ or -] 0.1 (d)
20:3n-6                            --                        --
20:4n-6                  3.5 [+ or -] 0.6         3.8 [+ or -] 0.3
22:4n-6                  9.0 [+ or -] 1.7         7.7 [+ or -] 0.4
22:5n-6                  0.7 [+ or -] 0.2 (b)     0.5 [+ or -] 0.l (b)
[SIGMA]PUFAn-6          14.7 [+ or -] 2.2 (b)    13.6 [+ or -] 0.5 (b)
[SIGMA]polyunsaturates  49.4 [+ or -] 3.0 (b)    50.4 [+ or -] 1.8 (b)
16:0DMA                  1.9 [+ or -] 0.2 (b)     1.4 [+ or -] 0.1 (c)
18:0DMA                  8.1 [+ or -] 0.6 (b)     9.3 [+ or -] 0.3 (c)

Values are mean [+ or -] S.D. n = number of samples.
Within a row, different superscript letters represent
significant differences (p < 0.05).

TABLE 3.
Fatty acid composition (as % of total
fatty acids) of veligers from
P. aurantiomarginata and B. columbina.

Fatty acids              P. aurantiomarginata        B. columbina
                               (n = 19)                 (n = 9)

14:0                     1.2 [+ or -] 0.6 (a)      0.6 [+ or -] 0.2
15:0                       1.3 [+ or -] 0.2        0.6 [+ or -] 0.2
16:0                     13.3 [+ or -] 1.7 (a)     17.3 [+ or -] 1.2
17:0                     5.3 [+ or -] 1.1 (a)      2.6 [+ or -] 0.2
18:0                     10.2 [+ or -] 0.9 (a)   5.2 [+ or -] 0.3 (a)
[SIGMA]saturates         31.4 [+ or -] 2.3 (a)     26.4 [+ or -] 1.5
16:1n-7                    2.4 [+ or -] 1.4      2.0 [+ or -] 0.5 (a)
18:1n-7                  2.8 [+ or -] 0.5 (a)    1.6 [+ or -] 0.2 (a)
18:1n-9                  3.8 [+ or -] 1.0 (a)      4.7 [+ or -] 0.6
20:1n-7                  0.9 [+ or -] 0.2 (a)      2.7 [+ or -] 0.4
20:1 n-9                 1.8 [+ or -] 0.3 (a)    0.7 [+ or -] 0.2 (a)
22:1n-9                           --               0.4 [+ or -] 0.2
[SIGMA]monounsaturates     11.7 [+ or -] 2.2     12.1 [+ or -] 0.8 (a)
18:3n-3                    0.7 [+ or -] 0.3        0.5 [+ or -] 0.3
20:3n-3                  0.8 [+ or -] 0.1 (a)             --
20:4n-3                    0.7 [+ or -] 0.2        0.2 [+ or -] 0.1
20:5n-3                    8.5 [+ or -] 0.8        9.1 [+ or -] 1.3
22:5n-3                  2.8 [+ or -] 0.6 (a)    7.4 [+ or -] 0.7 (a)
22:6n-3                    20.2 [+ or -] 2.8     12.6 [+ or -] 1.1 (a)
[SIGMA]PUFAn-3             33.8 [+ or -] 3.9     29.9 [+ or -] 2.3 (a)
16:2n-4                  0.8 [+ or -] 0.2 (a)    2.2 [+ or -] 0.3 (a)
16:3n-4                  0.4 [+ or -] 0.2 (a)    0.3 [+ or -] 0.1 (a)
[SIGMA]PUFAn-4           1.3 [+ or -] 0.3 (a)      2.5 [+ or -] 0.3
18:2n-6                  0.7 [+ or -] 0.1 (a)    0.4 [+ or -] 0.1 (a)
18:3n-6                    0.3 [+ or -] 0.1        0.3 [+ or -] 0.1
20:2n-6                    1.4 [+ or -] 0.21       0.5 [+ or -] 0.1
20:3n-6                  0.3 [+ or -] 0.1 (a)             --
20:4n-6                    4.0 [+ or -] 1.2      4.0 [+ or -] 0.3 (a)
22:4n-6                  4.1 [+ or -] 0.7 (a)    10.8 [+ or -] 1.0 (a)
22:5n-6                    1.6 [+ or -] 0.5      1.6 [+ or -] 0.5 (a)
[SIGMA]PUFAn-6           12.3 [+ or -] 1.9 (a)   17.7 [+ or -] 1.3 (a)
[SIGMA]polyunsaturates   47.4 [+ or -] 0.2 (a)     50.2 [+ or -] 1.8
16:0DMA                    2.6 [+ or -] 0.2        1.7 [+ or -] 0.1
18:0DMA                  6.9 [+ or -] 0.7 (a)    9.6 [+ or -] 1.0 (a)

Values are mean [+ or -] S.D. n = number of samples. a: P < 0.05 with
respect to the corresponding egg fatty acid value.