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The Chilean scallop Argopecten purpuratus (Lamarck, 1819): I. fatty acid composition and lipid content of six organs
Comparative Biochemistry and Physiology Part B 123 (1999) 89 – 96
The Chilean scallop Argopecten purpuratus (Lamarck, 1819): I. fatty acid composition and lipid content of six organs Marrit Caers a,*, Peter Coutteau a, Karen Cure a, Veronica Morales b, Gonzalo Gajardo b, Patrick Sorgeloos a b
a Laboratory of Aquaculture & Artemia Reference Center, Uni6ersity of Ghent, Rozier 44, B-9000 Gent, Belgium Uni6ersidad de Los Lagos, Department of Basic Sciences, Laboratory of Genetics and Aquaculture, P.O. Box 933, Osorno, Chile
Received 24 July 1998; received in revised form 10 February 1999; accepted 3 March 1999
Abstract This paper describes the distribution of lipids and fatty acids in different organs of Argopecten purpuratus broodstock. The female gonad and the digestive gland showed the highest lipid content, moderate lipid levels were present in the gills and male gonad while the mantle and especially the adductor muscle exhibited the lowest lipid content. A principal component analysis of the fatty acids of the total lipids separated the organs in four major groups: gills and mantle (I), adductor and male gonad (II), female gonad (III) and digestive gland (IV). A special feature of the gills and mantle was the presence of high levels of plasmalogens recognized by the peaks for vinyl methyl ethers from dimethylacetal degradation accompanying peaks for methyl esters of fatty acids in the GC profiles, and an unidentified fatty acid (22?). The highest level of n-3 polyunsaturated fatty acids (mainly EPA and DHA) was found in the adductor. Similarities between the fatty acid composition of the triglyceride fraction of the female gonad and the digestive gland (e.g. the high level of 14:0 and 18:4n-3) indicated the transfer of lipids from the lipid-rich digestive gland to the female gonad. Trimethyltridecanoic acid (TMTD) was found nearly exclusively in the polar lipid fraction of the digestive gland. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Bivalve; Scallop; Lipid; Fatty acid; Argopecten purpuratus
1. Introduction The Chilean scallop Argopecten purpuratus (Lamarck 1819), inhabits sedimentary substrates in sheltered embayments along the coast of the Pacific Ocean from the Gulf of Arauco, Chile (37° 10%S) to Paracas Bay, Peru (13° 50%S) [26,45]. A. purpuratus is a functional hermaphrodite mainly found in northern Chile, where it is reproductive all year, with spawning peaks occurring in late summer and autumn [26,44]. In southern Chile, where the species does not occur naturally but has been transplanted for colonization or aquaculture purposes, the gonads are mature from spring to autumn and are * Corresponding author. Tel.: +32-9-2643754; fax: 2644193. E-mail address: [email protected] (M. Caers)
+32-9-
recessive in the winter months [41]. A. purpuratus used to be an ecologically-important member of the subtidal macro-invertebrate assemblages along the northern part of the Chilean coast, particularly in Tongoy Bay [45]. This was the most important natural bed before this species became a valuable food product both for the internal and international market [10,26]. A severe drop in mollusc landings due to overfishing encouraged the aquaculture production of A. purpuratus [10]. Presently, mainly cultured animals account for production figures of this species [33]. However, production greatly depends on natural spat collection which is limited and highly unpredictable [10]. Despite significant improvements in hatchery facilities and culture technology, the main bottleneck for upscaling production remains the limited biological knowledge with regard to the nutri-
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tional requirements and feeding regimes in broodstock and early stages. Information concerning the fatty acid composition of A. purpuratus barely exists and is limited to fatty acid analysis of the total lipids either in the whole animal or in the gonad (male and female analyzed together) [22]. Bivalve lipid and fatty acid studies often focus either on the composition of the whole body [3,4,29] or major tissue groups [30,38], whereas little attention has been given to the analysis of individual organs. This paper describes the distribution of fatty acids in different organs in broodstock of A. purpuratus as an initial step to provide base-line data for further characterization of the fatty acid requirements in this and other bivalve species. This will help to understand the specific role of lipids and fatty acids in various organs as well as to evaluate their role in reproduction and other vital biological activities such as the provision of energy and essential fatty acids.
2. Materials and methods
2.1. Data collection Three-year-old scallops (live weight= 44.2 9 6.2 g), provided by the company ‘Salmones huito’ (Calbuco, region X, Chile), were transferred to the laboratory facilities near Puerto Montt in April (Autumn). The animals were cleaned and induced to spawn by thermal stimulation (none of the animals spawned). They were held in 1-mm filtered and UV-treated seawater at a temperature of 16°C for 48 h to clear their gut. After removal of the shells, the scallops was thoroughly rinsed with tap water to remove salts, blotted dry on paper towel covered with nytex netting and weighed (meat wet weight = 30.8 9 3.9 g). Gills, mantle, adductor muscle, digestive gland and gonads (separated into male and female part) were removed and the wet weight (WW) of each individual organ was determined. Samples were stored at − 20°C. Lipid and fatty acid analyses were performed on freeze-dried organs (three samples, each containing the organs of three scallops). For each sample, triplicate dry weight (DW; 24 h at 60°C) and ash (24 h at 450°C) determinations were performed. The organic matter content (OM) was calculated as the difference between DW and ash weight and expressed as a percentage of the DW.
2.2. Total lipid extraction and quantification Total lipids were extracted with the solvent mixture chloroform: methanol (2:1, v/v) [8]. Following solvent evaporation under nitrogen, lipids were transferred with three times 0.5 ml solvent mixture into a pre-weighed 2 ml vial. The solvent mixture was again evaporated
under nitrogen and the extracts were further dried overnight in a vacuum desiccator. After weighing, lipids were redissolved in the solvent mixture at a concentration of 10 mg/ml and 0.01% butylated hydroxy toluene (BHT) was added as an antioxidant.
2.3. Fatty acid analysis and quantification Fatty acid methyl esters (FAME) of total lipid were prepared by transmethylation with a mixture of sulfuric acid and methanol (1:100, v/v) for 16 h at 50°C [8]. FAME were analyzed with a Chrompack CP9001 gas chromatograph as described in Caers et al. [6]. During acid-catalyzed transmethylation, FAME are formed simultaneously with dimethylacetals (DMA) which originate from the 1-alkenyl chains of plasmalogens [8]. One sample of the methyl ester product from each organ was purified by HPTLC using toluene as the solvent system [8]. The FAME and DMA (which migrate in front of the FAME) bands were scraped off and extracted and analyzed separately as described above for FAME to identify DMA peaks. In routine samples, FAME and DMA were analyzed together. Therefore, the level of individual DMA and FAME represented in the graphs and tables, are expressed as percentage of the sum of all DMA and FAME present in the sample. Individual FAME and DMA were identified by reference to authentic standards (Nu-Chek Prep., USA) and well-characterized Pecten maximus samples provided by Soudant et al. [36]. Samples and standards were also injected into a gas chromatograph (Carlo Erba) equipped with a non-polar column. Comparison of A. purpuratus FAME extracts with well-characterized P. maximus samples provided by Soudant [35] revealed that an unidentified fatty acid which was mainly found in the gills and mantle of A. purpuratus corresponded with an unidentified fatty acids in the gonads of P. maximus. Since this fatty acid was represented in the work of Soudant et al. [36] as 22?, we use the same annotation in this study. 22? has tentatively been identified as a 22:4n-9 isomer, however not in the usual cis configuration but further research is needed to determine its exact structure [35]. Integrations and calculations were done with the software program ‘Maestro’ (Chrompack). For the preparation of FAME and DMA of the polar lipids (PL) and triglycerides (TAG), 3 mg of lipid were separated on TLC plates (20x20 cm, Silica gel 60, Merck, Germany) using hexane:diethyl ether:acetic acid (80:20:2,v/v) as developing solvent. A standard mixture was spotted alongside. Lipid classes were visualized under UV light after spraying with 0.1% 2%-7% dichlorofluorescein in 97% ethanol. The standard mixture run on the same plate was also sprayed with 4% iodine (I2) in methanol [8,11]. The polar lipids and the triglycerides
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were scraped off and transmethylated as described for the total lipids. Finally, the whole plate was sprayed with 4% I2 in methanol to make sure the bands were scraped off properly.
2.4. Statistical analysis Statistical analysis of the data included one-way ANOVA’s, Tukey Honest Significant Differences Test (HSD) and principal component analysis and was performed with the software program STATISTICA (Microsoft, StatSoft, Inc.). The homogeneity of the variances of means was checked by the Univariate Test (Cohran C, Hartley, Barlett). Arcsine transformation was used prior to statistical analyses for data expressed in percentages [34].
3. Results Table 1 displays the wet weight, organic matter content and total lipid content for six organs analyzed. The average gonad index of A. purpuratus, defined as the percentage of shell-free body WW that consisted of gonad WW (male and female together) [22] was 8.0%. The organic matter content of the gills and mantle was significantly lower than in the other organs. The female gonad and the digestive gland showed the highest lipid content, moderate lipid levels were present in the gills and male gonad while the mantle and especially the adductor muscle exhibited the lowest lipid content. Table 2 shows the fatty acid composition of each organ. Irrespective of the organ, the total n-3 polyunsaturated fatty acids (PUFA) were the most important fatty acid type and were dominated by docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n3, EPA), followed by the total saturated fatty acids Table 1 WW (% of shell-free body WW), OM (% of DW) and total lipid content (TL; mg/g DW and mg/g OM) of six organs of the Chilean scallop A. purpuratus a Tissueb
WW (%)
OM (%)
TL (mg/g DW)
TL (mg/g OM)
Gi Ma Ad Dg FGo MGo
9.3 91.4a 21.2 91.9b 7.5 90.4a 36.1 96.0c 8.0 91.1ac
81.99 1.4a 80.59 0.6b 93.29 0.6c 93.29 0.1c 90.39 0.7d 90.29 0.5d
77.691.3c 53.4 9 4.6b 32.79 3.8a 287.6 91.2f 190.7 92.4e 94.49 3.6d
94.892.3c 66.3 94.1b 35.093.4a 308.691.0f 212.0 9 2.4e 105.093.6d
a Data represent the average 9SD of three replicate samples, values with the same letter in one column are not significantly different (ANOVA, Tukey HSD test, PB0.5). b Ad, Adductor muscle; Dg, digestive gland; FGo, female gonad; Gi, gills; Ma, mantle; MGo, male gonad. c Whole gonad containing female and male part.
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(SAFA), principally 16:0. The total monounsaturated fatty acids (MUFA) levels were at least twice as high as the n-6 PUFA and were mainly represented by 16:1n-7 and C-18 and C-20 MUFA of the n-7 and n-9 families. The results of a principal component analysis are shown in Figs. 1 and 2. It was based on the individual fatty acids and DMA listed in Table 2 for the total lipids of the six organs. The first component, accounting for 55% of the total sample variance, separated the organs into four major groups, i.e. the gills and mantle (I), the adductor and male gonad (II), the female gonad (III) and the digestive gland (IV). The second component, accounting for 20% of the total sample variance, confirmed the existence of these four groups but further separated the individual organs in group I as well as further distancing group II and III from group I and IV. The corresponding factor loadings, plotted in Fig. 2, illustrate which fatty acids are contributing the most to segregate groups along the first and second principal axis. The numbers correspond with the number of each fatty acid and DMA in Table 2. The fatty acids in groups A and B distinguished the gills and mantle (group I) from groups II, III and IV, those of group C were characteristic for the male gonad or the adductor (group II). The abundance of each fatty acid of group D was typical for the digestive gland and the female gonad (groups III and IV), while the high levels of the group E fatty acids separated the female gonad (III) from the digestive gland (IV). As shown in Fig. 1, the fatty acid composition of the gills and mantle exhibited a similar profile though the DHA and EPA levels were significantly higher in the mantle than in the gills (Table 2). Both organs had the highest level of the two major n-6 PUFA, namely 20:4n-6 and 22:5n-6 (Fig. 2 and Table 2). However, most striking were the very high DMA levels, mainly 18:0DMA and 20:1DMA, and the abundance of an unidentified fatty acid (22:?) which clearly distinguished these organs from all the others. This fatty acid, which was always present at levels less than 3% in all the other organs, accounted for 9.2 and 7.6% in the gills and mantle, respectively (Figs. 1 and 2 and Table 2). Although the fatty acid profile of the adductor was very similar to that of the male gonad, there were also marked differences. The abundance of DHA and EPA in the adductor muscle yielded the highest total n-3 PUFA level among all organs while the male gonad was the only organ showing a relatively high level of 22:5n-3. Among all organs, the male gonad showed the highest level of 16:0 and 20:1n-7 whereas the highest 18:1n-7 and 20:2n-6 level was detected in the adductor. The principal component analysis clearly discriminated between the fatty acid composition of the male and female gonad. The level of 14:0 was more than twice as high in the female gonad, though a significantly lower 16:0 and 18:0 level diminished the SAFA
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Table 2 Fatty acid composition (%) of various organsa of the Chilean scallop A. purpuratus b No
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Fatty acid and DMA
TMTD 12:0 14:0 16:0 18:0 SAFA 16:1n-7 18:1n-9 18:1n-7 20:1n-9 20:1n-7 MUFA 18:2n-6 20:2n-6 20:4n-6 22:5n-6 n-6 PUFA 18:3n-3 18:4n-3 20:4n-3 20:5n-3 21:5n-3 22:5n-3 22:6n-3 n-3 PUFA 22:? 16:0DMA 17:0DMA 18:0DMA 20:1DMA total DMA n-3/n-6 DHA/EPA
Gi TLc
trd 2.0a 3.1c 14.0c 6.2a 27.2b 2.6c 2.1cd 1.6c 2.8a 1.3b 11.6d 0.6c 0.8c 1.7a 0.9a 4.9a 0.8c 1.2d tr 8.1d tr 1.0c 16.0b 28.3f 9.2a 1.2b 0.8a 9.2a 4.2a 16.8a 6.0d 2.0b
Ma TL
nd tr 2.5d 14.4c 6.6a 25.2c 1.6d 1.8d 2.1b 1.7bc 0.7e 9.1e tr 0.8bc 1.9a 0.8a 4.4b 0.7c 1.2d tr 9.5c tr 0.8c 23.7a 37.0e 7.6b 1.7a tr 7.0b 3.1b 12.7b 8.6c 2.5a
Ad TL
nd tr 2.1e 18.9b 4.9b 27.3b 1.1e 1.2e 2.6a 2.2b 0.8cd 9.2e tr 1.4a 1.0b 0.6b 4.1b 0.9bc 2.1c 1.6a 16.4a 0.9a 1.6b 25.6a 49.4a 2.6c tr tr 3.0c 1.3c 5.2d 12.1b 1.6c
MGo TL
nd 1.6b 2.7cd 21.8a 6.1a 33.7a 2.3c 2.4c 1.7bc 2.9a 2.3a 13.1c 0.9b 1.0b 0.7c tr 3.5c 1.0b 2.0c 1.6a 14.3b 0.9a 4.4a 14.3c 39.4d 2.4c 0.9c nd 3.3c 1.4c 5.9c 11.3b 1.0d
FGo TL
tr tr 4.2b 17.9b 3.2c 26.5bc 6.6b 4.3a 1.9bc 1.7cd 1.0c 16.8b 1.3a 0.8bc 0.6c tr 3.7c 2.1a 5.7b 1.0c 18.0a 0.9a 0.8c 14.2c 43.2b 2.3c tr tr 1.8d 0.9d 3.2e 11.9b 0.8d
Dg TL
2.6a tr 7.0a 14.3c 3.4c 25.7bc 8.2a 3.5b 1.9bc 1.3d 0.7de 19.8a 1.4a 0.5d tr tr 3.0d 2.4a 8.7a 1.3b 17.7a 1.0a tr 9.2d 41.3c 1.2d tr tr 0.8e tr 1.4f 14.1a 0.5e
FGo
Dg
PL
TAG
PL
TAG
tr nd 1.8D 13.8B 5.3B 21.9C 0.8C 1.9D 1.2C tr 1.3A 7.9C tr tr 1.5 0.9 3.6B 0.7C 2.2D tr 13.1B 0.8A 1.4A 22.3A 41.0B 4.9A 1.7A tr 10.9A 5.6A 18.9A 11.6B 1.7A
tr nd 5.8B 21.2A 1.9D 29.7A 8.3A 5.0A 1.9B 2.0A 0.9B 19.4A 1.4B 1.0A tr tr 3.6B 2.5A 6.6B 1.2A 18.4A 1.0A tr 11.0C 42.0B 1.6C nd tr 0.7C nd 1.0C 11.9B 0.6C
6.8 tr 4.1C 14.7B 6.8A 26.8B 4.6B 3.0C 1.2C 1.0C tr 12.1B 1.1C tr tr tr 2.3C 1.3B 3.6C tr 12.2B tr 1.0B 14.4B 33.1C 3.5B 1.4A tr 7.9B 2.8B 12.5B 14.4A 1.2B
tr nd 7.7A 14.8B 2.4C 25.8B 9.5A 4.1B 2.4A 1.4B 1.2AB 19.4A 1.9A 0.7B tr tr 4.0A 2.9A 9.8A 1.1A 20.3A 1.0A 0.6C 9.4D 46.0A tr nd tr 0.8C nd 1.2C 11.6B 0.5C
a
Abbreviations as in Table 1. Data represent the average of three replicate samples. Values for the TL of the six organs with the same letter in one row are not significantly different, values for the PL and TAG fraction of the digestive gland and female gonad with the same capital letter in one row are not significantly different (ANOVA, Tukey HSD, PB0.5). c Total lipid. d Trace (50.5%). b
in female more significantly than in the male gonad. Compared to the male gonad, the proportions of the MUFA, respectively, n-3 PUFA were significantly higher in the female gonad mainly due to higher 16:1n7 and 18:1n-9, 18:3n-3, 18:4n-3 and 20:5n-3 levels, respectively. The fatty acids which distinguished the female gonad from groups I and II were even more abundant in the digestive gland (e.g. the 18:4n-3 and 14:0 levels reached 8.7 and 7.0%, respectively). Apart from the similarities between the digestive gland and the female gonad, the abundance of trimethyltridecanoic acid (TMTD) and 14:0 separated the digestive gland (IV) from the female gonad (III). Separate analysis of the PL and triglycerides (TAG) of the latter organs illustrated that the
high levels of 14:0, 16:1n-7, 18:1n-9, 18:3n-3, 18:4n-3 and EPA in the total lipids of the female gonad and digestive gland originated from the TAG fraction. The TAG of both organs contained more EPA but less DHA than the PL fraction. In both organs, DMA were found nearly exclusively in the PL.
4. Discussion The lower organic matter content of the gills and mantle was in agreement with data for other scallop species such as Chlamys septemradiata [1] and Chlamys islandica [37]. However, there is little information available to compare the results since most data on ash
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Fig. 1. Principal component analysis based on the fatty acids in the total lipids of six organs of the Chilean scallop A. purpuratus. The first and second principal component are plotted in an x– y plane and accounted for 55 and 20%, respectively, of the total sample variance. Group I includes the gills (hollow circle) and mantle (filled circle), group II includes the male gonad (hollow triangle) and the adductor (filled triangle) and group III and IV are formed by the female gonad (hollow rectangular) and digestive gland (filled rectangular), respectively.
content refer to the whole body tissue and are often expressed as % of the total wet weight [5]. The lipid content of the mantle of A. purpuratus (5.3% of the DW) was lower than in the clam Macoma baltica (mantle+siphon, 7.4%) [12] or the mussel Mytilus edulis (9–14%) [46]. Compared to the values reported by Nevenzel et al. [27] for the oyster Crassostrea 6irginica (15.5%) and especially by Klingensmith and Stillway [17] for the hardshell clam Mercenaria mercenaria (26.3–26.5%), quite low lipid concentrations were found in the gills of A. purpuratus (present study) and M. edulis (7–8%) [46]. Values reported in the literature for the lipid content of the gills, expressed as percentage of wet weight, also vary widely, e.g. from 0.6% in the mussel Mytilus californianus and 0.8% in the scallops Placopecten magellanicus and Hinites multirugosus to 1.7% in the mussel Mytilus gallopro6incialis [20,24,27]. In agreement with the fatty acid profile of the gills and mantle of the sea scallop P. magellanicus [25], the EPA levels in the gills and mantle of A. purpuratus were significantly lower than in the other organs. The unidentified fatty acid (22:?) found in A. purpuratus has previously be found in the gonads and larvae of Pecten maximus [23,36] but always at low levels, comparable with the levels found in this study for the gonads, adductor muscle and digestive gland. The very high levels found in the gills and mantle have not been reported earlier and suggest that 22:? has a unique function in these organs although information on its origin and metabolism are completely lacking.
Interestingly, this fatty acid was not present in the gills of the clam M. mercenaria or the scallop P. magellanicus which were both characterized by high levels of 20:1 fatty acid isomers [18,25]. In A. purpuratus, the highest 20:1 levels were detected in the male gonad, followed by the gill, but they were considerably lower than those reported by the latter authors. High 20:1 levels were also reported by Zhukova [47] in the polar lipids of the gills and mantle of M. edulis (12.1 and 10.3%, respectively) together with very high levels of non-methyleneinterrupted dienoic fatty acids (NMID) especially 20:2. Pectinidae normally contains no or only trace levels of NMID [13,14,47]. Although an adequate explanation is not available at present, the results indicate that the 20:1 isomers and NMID are replaced by 22:? in the gills and mantle of A. purpuratus. Other examples of fatty acid profiles of bivalve gills that are remarkably different from that of most other bivalve species can be found in Piretti et al. [30] and Klingensmith [18] for the clams Scapharca inaequi6al6is (12.5% 17:0) and M. mercenaria (26.3 and 44.9% 16:0 and 15.2 and 16.3% 18:0 in the polar and neutral lipids, respectively, and less than 1% DHA in both lipid fractions) and in Fouad et al. [9] for the mussel Mytilus gallopro6incialis (7.6% 18:4n-3, 6.7% 22:1n-9 and 6.3% 16:4). The latter results are distinctly different from the fatty acid profile of other clams and mussels reported in the reviews of Joseph [13,14]. Consistent with available data in literature, the adductor muscle exhibited a low lipid content compared
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Fig. 2. The plot of the factor loadings shows which and how much each fatty acid contributed to the dispersion of the four groups (I, II, III, IV) in Fig. 1. Each fatty acid group (A, B, C, D and E) includes fatty acids which distinguished the individual groups in Fig. 1 (see text for more details). The numbers correspond with the number of each fatty acid and DMA in Table 2.
to the other major organs since glycogen is the main storage form and the fatty acid profile is characterized by a high n-3 PUFA level [1,12,17,24,25]. On the contrary, in M. mercenaria, the lowest n-3 PUFA level was detected in the adductor [18]. In several bivalve species, including A. purpuratus [21], the development of the gonad is associated with a decrease of the carbohydrate reserves in the adductor muscle as glycogen reserves can be used for de novo synthesis of lipids which accumulate in the female gonad during maturation. Similar to other Pectinidae [1,24,39], the lipid level in the male gonad was significantly lower than in the female gonad. Data on the male gonad are scarce compared to the comprehensive information available for the female gonad, and in case of hermaphrodite species, the male and female part are commonly analyzed together [13,22]. The results of studies which allow the fatty acid profiles of the male and female gonad to be compared [25,36] do not yield a uniform trend with respect to the levels of the major fatty acids or fatty acid groups, although 16:0, 20:5n-3 and 22:6n-3 seemed to be always abundant in both organs. The abundance of the group D fatty acids (14:0, 16:1n-7, 18:1n-9, 18:2n-6, 18:3n-3, 18:4n-3) in the TAG fraction of the female gonad and digestive gland clearly distinguished the latter organs from the others (Figs. 1 and 2). The specific anatomical distribution of these fatty acids is associated with the transfer of lipids from the lipid-rich digestive gland to the developing female gonad. This has been observed by several authors who
studied the seasonal lipid or fatty acid composition of both organs [3,25,38,46] and it was also confirmed in 14 C studies with the scallops Chlamys hericia [43] and A. irradiance [2]. On the other hand, Uriarte et al. [41] suggested that the digestive gland is not a storage organ in A. purpuratus since the condition index, defined as (DW organ*100)/(DW total meat), of the digestive gland was very constant during the year whereas the condition index of the adductor and the soft parts (gills+mantle) was negatively correlated with the gonad index. It is well known that during vitellogenesis, large amounts of neutral lipids are accumulating in developing eggs since they provide a major source of energy during embryogenesis [42]. This explains the severe drop of the lipid level in the female gonad of spawned animals [28,40]. TMTD originates from the degradation of dietary chlorophyll. Usually it is only found in substantial amounts in the digestive gland suggesting that it is quickly and efficiently catabolized in the digestive gland before it can be transported to other organs [13,14]. As in the present study, it is sometimes detected at low levels in other organs [13,16,25,30]. In the digestive gland of the scallops P. magellanicus, TMTD was mainly found in the TAG fraction [25]. Due to its characteristic anatomical distribution, the latter authors suggested that it could be a useful indicator of an autotrophic source of reduced carbon and of the nutritional status of the animal through estimation of the TAG reserves. The present study does not support this hypothesis as TMTD was mainly located in the PL and
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not in the TAG fraction of the digestive gland of A. purpuratus. Ether-containing phosphoglycerides are found in nearly all animal cells. In aquatic animals, highest plasmalogen concentrations are found in the gills, nerve and brain tissue. Their functional role in biological systems is not clearly understood, but available information suggest they may play an important function in membrane integrity [7]. Their abundance (detected as DMA) in the gills and mantle of A. purpuratus, two organs which have a great surface in direct contact with the water, appears to add support to this view. Rapport [31] and Nevenzel et al. [27] have previously reported the abundance of plasmalogen in bivalve gill lipids. In contrast with other marine organisms, the DMA composition of bivalves is very poorly documented and none of these studies compared the qualitative or quantitative differences in the DMA profile of different bivalve organs [15,19,32].
Acknowledgements This work has been funded in part by grants from the Flemish government, IWT (Marrit Caers) and the European Union (contract IC18-CT97-0188). We are also grateful to Fondecyt (grant 1970807) and the FONDAP-OMB study group for support as well as the company ‘Salmones Huito’ (Calbuco) for supplying the scallops. We wish to thank P. Soudant, Y. Marty and J.F. Samain for providing well-identified P. maximus FAME extracts.
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[29] Piretti MV, Taioli F, Pagliuca G. Investigation of the seasonal variations of sterol and fatty acid constituents in the bivalve molluscs Venus gallina and Scapharca inaequi6al6is (Bruguiere). Comp Biochem Physiol 1987;88B:1201–8. [30] Piretti MV, Zuppa F, Pagliuca G, Taioli F. Investigation of the seasonal variations of fatty acid constituents in selected tissues of the bivalve mollusc Scapharca inaequi6al6is (Bruguiere). Comp Biochem Physiol 1988;89B:183–7. [31] Rapport MM. The a, b-unsaturated ether (plasmalogen) content of the tissues of several mollusks. Biol Bull 1961;121:376 – 7. [32] Sargent JR. Ether-linked glycerides in marine animals. In: Ackman RG, editor. Marine Biogenic Lipids, Fats and Oils. Boca Raton, FL: CRC Press, 1989:175–97. [33] Sernap. Anuario Estadistico de Pesca 1996. Republica de Chile. Servicio National de Pesca. Chile, PA: Ministerio de Economica Fomento y Reconstruccion. Chile, 1996. [34] Sokal RR, Rohlf FJ. Biometry. Principles and Practice of Statistics in Biological Research. San Francisco, CA: W.H. Freeman, 1995. [35] Soudant P. Les phospholipides et les sterols des geniteurs et des larves de coquille Saint Jacques Pecten maximus (L.). Relations avec la nutrition. The`se de Doctorat de l’Universite´ de Brest, 1995. [36] Soudant P, Marty Y, Moal J, Robert R, Quere C, Le Coz JR, Samain JF. Effect of food fatty acid and sterol quality on Pecten maximus gonad composition and reproduction process. Aquaculture 1996;143:361 – 78. [37] Sundet JH, Vahl O. Seasonal changes in dry weight and biochemical composition of the tissues of sexually mature and immature Iceland scallops, Chlamys islandica. J Mar Biol Assoc UK 1981;61:1001 – 10. [38] Taylor AC, Venn TJ. Seasonal variation in weight and biochemical composition of the tissues of the queen scallop, Chlamys
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The Chilean scallop Argopecten purpuratus (Lamarck, 1819): I. fatty acid composition and lipid content of six organs Marrit Caers a,*, Peter Coutteau a, Karen Cure a, Veronica Morales b, Gonzalo Gajardo b, Patrick Sorgeloos a b
a Laboratory of Aquaculture & Artemia Reference Center, Uni6ersity of Ghent, Rozier 44, B-9000 Gent, Belgium Uni6ersidad de Los Lagos, Department of Basic Sciences, Laboratory of Genetics and Aquaculture, P.O. Box 933, Osorno, Chile
Received 24 July 1998; received in revised form 10 February 1999; accepted 3 March 1999
Abstract This paper describes the distribution of lipids and fatty acids in different organs of Argopecten purpuratus broodstock. The female gonad and the digestive gland showed the highest lipid content, moderate lipid levels were present in the gills and male gonad while the mantle and especially the adductor muscle exhibited the lowest lipid content. A principal component analysis of the fatty acids of the total lipids separated the organs in four major groups: gills and mantle (I), adductor and male gonad (II), female gonad (III) and digestive gland (IV). A special feature of the gills and mantle was the presence of high levels of plasmalogens recognized by the peaks for vinyl methyl ethers from dimethylacetal degradation accompanying peaks for methyl esters of fatty acids in the GC profiles, and an unidentified fatty acid (22?). The highest level of n-3 polyunsaturated fatty acids (mainly EPA and DHA) was found in the adductor. Similarities between the fatty acid composition of the triglyceride fraction of the female gonad and the digestive gland (e.g. the high level of 14:0 and 18:4n-3) indicated the transfer of lipids from the lipid-rich digestive gland to the female gonad. Trimethyltridecanoic acid (TMTD) was found nearly exclusively in the polar lipid fraction of the digestive gland. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Bivalve; Scallop; Lipid; Fatty acid; Argopecten purpuratus
1. Introduction The Chilean scallop Argopecten purpuratus (Lamarck 1819), inhabits sedimentary substrates in sheltered embayments along the coast of the Pacific Ocean from the Gulf of Arauco, Chile (37° 10%S) to Paracas Bay, Peru (13° 50%S) [26,45]. A. purpuratus is a functional hermaphrodite mainly found in northern Chile, where it is reproductive all year, with spawning peaks occurring in late summer and autumn [26,44]. In southern Chile, where the species does not occur naturally but has been transplanted for colonization or aquaculture purposes, the gonads are mature from spring to autumn and are * Corresponding author. Tel.: +32-9-2643754; fax: 2644193. E-mail address: [email protected] (M. Caers)
+32-9-
recessive in the winter months [41]. A. purpuratus used to be an ecologically-important member of the subtidal macro-invertebrate assemblages along the northern part of the Chilean coast, particularly in Tongoy Bay [45]. This was the most important natural bed before this species became a valuable food product both for the internal and international market [10,26]. A severe drop in mollusc landings due to overfishing encouraged the aquaculture production of A. purpuratus [10]. Presently, mainly cultured animals account for production figures of this species [33]. However, production greatly depends on natural spat collection which is limited and highly unpredictable [10]. Despite significant improvements in hatchery facilities and culture technology, the main bottleneck for upscaling production remains the limited biological knowledge with regard to the nutri-
0305-0491/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 3 0 5 - 0 4 9 1 ( 9 9 ) 0 0 0 4 6 - 2
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tional requirements and feeding regimes in broodstock and early stages. Information concerning the fatty acid composition of A. purpuratus barely exists and is limited to fatty acid analysis of the total lipids either in the whole animal or in the gonad (male and female analyzed together) [22]. Bivalve lipid and fatty acid studies often focus either on the composition of the whole body [3,4,29] or major tissue groups [30,38], whereas little attention has been given to the analysis of individual organs. This paper describes the distribution of fatty acids in different organs in broodstock of A. purpuratus as an initial step to provide base-line data for further characterization of the fatty acid requirements in this and other bivalve species. This will help to understand the specific role of lipids and fatty acids in various organs as well as to evaluate their role in reproduction and other vital biological activities such as the provision of energy and essential fatty acids.
2. Materials and methods
2.1. Data collection Three-year-old scallops (live weight= 44.2 9 6.2 g), provided by the company ‘Salmones huito’ (Calbuco, region X, Chile), were transferred to the laboratory facilities near Puerto Montt in April (Autumn). The animals were cleaned and induced to spawn by thermal stimulation (none of the animals spawned). They were held in 1-mm filtered and UV-treated seawater at a temperature of 16°C for 48 h to clear their gut. After removal of the shells, the scallops was thoroughly rinsed with tap water to remove salts, blotted dry on paper towel covered with nytex netting and weighed (meat wet weight = 30.8 9 3.9 g). Gills, mantle, adductor muscle, digestive gland and gonads (separated into male and female part) were removed and the wet weight (WW) of each individual organ was determined. Samples were stored at − 20°C. Lipid and fatty acid analyses were performed on freeze-dried organs (three samples, each containing the organs of three scallops). For each sample, triplicate dry weight (DW; 24 h at 60°C) and ash (24 h at 450°C) determinations were performed. The organic matter content (OM) was calculated as the difference between DW and ash weight and expressed as a percentage of the DW.
2.2. Total lipid extraction and quantification Total lipids were extracted with the solvent mixture chloroform: methanol (2:1, v/v) [8]. Following solvent evaporation under nitrogen, lipids were transferred with three times 0.5 ml solvent mixture into a pre-weighed 2 ml vial. The solvent mixture was again evaporated
under nitrogen and the extracts were further dried overnight in a vacuum desiccator. After weighing, lipids were redissolved in the solvent mixture at a concentration of 10 mg/ml and 0.01% butylated hydroxy toluene (BHT) was added as an antioxidant.
2.3. Fatty acid analysis and quantification Fatty acid methyl esters (FAME) of total lipid were prepared by transmethylation with a mixture of sulfuric acid and methanol (1:100, v/v) for 16 h at 50°C [8]. FAME were analyzed with a Chrompack CP9001 gas chromatograph as described in Caers et al. [6]. During acid-catalyzed transmethylation, FAME are formed simultaneously with dimethylacetals (DMA) which originate from the 1-alkenyl chains of plasmalogens [8]. One sample of the methyl ester product from each organ was purified by HPTLC using toluene as the solvent system [8]. The FAME and DMA (which migrate in front of the FAME) bands were scraped off and extracted and analyzed separately as described above for FAME to identify DMA peaks. In routine samples, FAME and DMA were analyzed together. Therefore, the level of individual DMA and FAME represented in the graphs and tables, are expressed as percentage of the sum of all DMA and FAME present in the sample. Individual FAME and DMA were identified by reference to authentic standards (Nu-Chek Prep., USA) and well-characterized Pecten maximus samples provided by Soudant et al. [36]. Samples and standards were also injected into a gas chromatograph (Carlo Erba) equipped with a non-polar column. Comparison of A. purpuratus FAME extracts with well-characterized P. maximus samples provided by Soudant [35] revealed that an unidentified fatty acid which was mainly found in the gills and mantle of A. purpuratus corresponded with an unidentified fatty acids in the gonads of P. maximus. Since this fatty acid was represented in the work of Soudant et al. [36] as 22?, we use the same annotation in this study. 22? has tentatively been identified as a 22:4n-9 isomer, however not in the usual cis configuration but further research is needed to determine its exact structure [35]. Integrations and calculations were done with the software program ‘Maestro’ (Chrompack). For the preparation of FAME and DMA of the polar lipids (PL) and triglycerides (TAG), 3 mg of lipid were separated on TLC plates (20x20 cm, Silica gel 60, Merck, Germany) using hexane:diethyl ether:acetic acid (80:20:2,v/v) as developing solvent. A standard mixture was spotted alongside. Lipid classes were visualized under UV light after spraying with 0.1% 2%-7% dichlorofluorescein in 97% ethanol. The standard mixture run on the same plate was also sprayed with 4% iodine (I2) in methanol [8,11]. The polar lipids and the triglycerides
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were scraped off and transmethylated as described for the total lipids. Finally, the whole plate was sprayed with 4% I2 in methanol to make sure the bands were scraped off properly.
2.4. Statistical analysis Statistical analysis of the data included one-way ANOVA’s, Tukey Honest Significant Differences Test (HSD) and principal component analysis and was performed with the software program STATISTICA (Microsoft, StatSoft, Inc.). The homogeneity of the variances of means was checked by the Univariate Test (Cohran C, Hartley, Barlett). Arcsine transformation was used prior to statistical analyses for data expressed in percentages [34].
3. Results Table 1 displays the wet weight, organic matter content and total lipid content for six organs analyzed. The average gonad index of A. purpuratus, defined as the percentage of shell-free body WW that consisted of gonad WW (male and female together) [22] was 8.0%. The organic matter content of the gills and mantle was significantly lower than in the other organs. The female gonad and the digestive gland showed the highest lipid content, moderate lipid levels were present in the gills and male gonad while the mantle and especially the adductor muscle exhibited the lowest lipid content. Table 2 shows the fatty acid composition of each organ. Irrespective of the organ, the total n-3 polyunsaturated fatty acids (PUFA) were the most important fatty acid type and were dominated by docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n3, EPA), followed by the total saturated fatty acids Table 1 WW (% of shell-free body WW), OM (% of DW) and total lipid content (TL; mg/g DW and mg/g OM) of six organs of the Chilean scallop A. purpuratus a Tissueb
WW (%)
OM (%)
TL (mg/g DW)
TL (mg/g OM)
Gi Ma Ad Dg FGo MGo
9.3 91.4a 21.2 91.9b 7.5 90.4a 36.1 96.0c 8.0 91.1ac
81.99 1.4a 80.59 0.6b 93.29 0.6c 93.29 0.1c 90.39 0.7d 90.29 0.5d
77.691.3c 53.4 9 4.6b 32.79 3.8a 287.6 91.2f 190.7 92.4e 94.49 3.6d
94.892.3c 66.3 94.1b 35.093.4a 308.691.0f 212.0 9 2.4e 105.093.6d
a Data represent the average 9SD of three replicate samples, values with the same letter in one column are not significantly different (ANOVA, Tukey HSD test, PB0.5). b Ad, Adductor muscle; Dg, digestive gland; FGo, female gonad; Gi, gills; Ma, mantle; MGo, male gonad. c Whole gonad containing female and male part.
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(SAFA), principally 16:0. The total monounsaturated fatty acids (MUFA) levels were at least twice as high as the n-6 PUFA and were mainly represented by 16:1n-7 and C-18 and C-20 MUFA of the n-7 and n-9 families. The results of a principal component analysis are shown in Figs. 1 and 2. It was based on the individual fatty acids and DMA listed in Table 2 for the total lipids of the six organs. The first component, accounting for 55% of the total sample variance, separated the organs into four major groups, i.e. the gills and mantle (I), the adductor and male gonad (II), the female gonad (III) and the digestive gland (IV). The second component, accounting for 20% of the total sample variance, confirmed the existence of these four groups but further separated the individual organs in group I as well as further distancing group II and III from group I and IV. The corresponding factor loadings, plotted in Fig. 2, illustrate which fatty acids are contributing the most to segregate groups along the first and second principal axis. The numbers correspond with the number of each fatty acid and DMA in Table 2. The fatty acids in groups A and B distinguished the gills and mantle (group I) from groups II, III and IV, those of group C were characteristic for the male gonad or the adductor (group II). The abundance of each fatty acid of group D was typical for the digestive gland and the female gonad (groups III and IV), while the high levels of the group E fatty acids separated the female gonad (III) from the digestive gland (IV). As shown in Fig. 1, the fatty acid composition of the gills and mantle exhibited a similar profile though the DHA and EPA levels were significantly higher in the mantle than in the gills (Table 2). Both organs had the highest level of the two major n-6 PUFA, namely 20:4n-6 and 22:5n-6 (Fig. 2 and Table 2). However, most striking were the very high DMA levels, mainly 18:0DMA and 20:1DMA, and the abundance of an unidentified fatty acid (22:?) which clearly distinguished these organs from all the others. This fatty acid, which was always present at levels less than 3% in all the other organs, accounted for 9.2 and 7.6% in the gills and mantle, respectively (Figs. 1 and 2 and Table 2). Although the fatty acid profile of the adductor was very similar to that of the male gonad, there were also marked differences. The abundance of DHA and EPA in the adductor muscle yielded the highest total n-3 PUFA level among all organs while the male gonad was the only organ showing a relatively high level of 22:5n-3. Among all organs, the male gonad showed the highest level of 16:0 and 20:1n-7 whereas the highest 18:1n-7 and 20:2n-6 level was detected in the adductor. The principal component analysis clearly discriminated between the fatty acid composition of the male and female gonad. The level of 14:0 was more than twice as high in the female gonad, though a significantly lower 16:0 and 18:0 level diminished the SAFA
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Table 2 Fatty acid composition (%) of various organsa of the Chilean scallop A. purpuratus b No
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Fatty acid and DMA
TMTD 12:0 14:0 16:0 18:0 SAFA 16:1n-7 18:1n-9 18:1n-7 20:1n-9 20:1n-7 MUFA 18:2n-6 20:2n-6 20:4n-6 22:5n-6 n-6 PUFA 18:3n-3 18:4n-3 20:4n-3 20:5n-3 21:5n-3 22:5n-3 22:6n-3 n-3 PUFA 22:? 16:0DMA 17:0DMA 18:0DMA 20:1DMA total DMA n-3/n-6 DHA/EPA
Gi TLc
trd 2.0a 3.1c 14.0c 6.2a 27.2b 2.6c 2.1cd 1.6c 2.8a 1.3b 11.6d 0.6c 0.8c 1.7a 0.9a 4.9a 0.8c 1.2d tr 8.1d tr 1.0c 16.0b 28.3f 9.2a 1.2b 0.8a 9.2a 4.2a 16.8a 6.0d 2.0b
Ma TL
nd tr 2.5d 14.4c 6.6a 25.2c 1.6d 1.8d 2.1b 1.7bc 0.7e 9.1e tr 0.8bc 1.9a 0.8a 4.4b 0.7c 1.2d tr 9.5c tr 0.8c 23.7a 37.0e 7.6b 1.7a tr 7.0b 3.1b 12.7b 8.6c 2.5a
Ad TL
nd tr 2.1e 18.9b 4.9b 27.3b 1.1e 1.2e 2.6a 2.2b 0.8cd 9.2e tr 1.4a 1.0b 0.6b 4.1b 0.9bc 2.1c 1.6a 16.4a 0.9a 1.6b 25.6a 49.4a 2.6c tr tr 3.0c 1.3c 5.2d 12.1b 1.6c
MGo TL
nd 1.6b 2.7cd 21.8a 6.1a 33.7a 2.3c 2.4c 1.7bc 2.9a 2.3a 13.1c 0.9b 1.0b 0.7c tr 3.5c 1.0b 2.0c 1.6a 14.3b 0.9a 4.4a 14.3c 39.4d 2.4c 0.9c nd 3.3c 1.4c 5.9c 11.3b 1.0d
FGo TL
tr tr 4.2b 17.9b 3.2c 26.5bc 6.6b 4.3a 1.9bc 1.7cd 1.0c 16.8b 1.3a 0.8bc 0.6c tr 3.7c 2.1a 5.7b 1.0c 18.0a 0.9a 0.8c 14.2c 43.2b 2.3c tr tr 1.8d 0.9d 3.2e 11.9b 0.8d
Dg TL
2.6a tr 7.0a 14.3c 3.4c 25.7bc 8.2a 3.5b 1.9bc 1.3d 0.7de 19.8a 1.4a 0.5d tr tr 3.0d 2.4a 8.7a 1.3b 17.7a 1.0a tr 9.2d 41.3c 1.2d tr tr 0.8e tr 1.4f 14.1a 0.5e
FGo
Dg
PL
TAG
PL
TAG
tr nd 1.8D 13.8B 5.3B 21.9C 0.8C 1.9D 1.2C tr 1.3A 7.9C tr tr 1.5 0.9 3.6B 0.7C 2.2D tr 13.1B 0.8A 1.4A 22.3A 41.0B 4.9A 1.7A tr 10.9A 5.6A 18.9A 11.6B 1.7A
tr nd 5.8B 21.2A 1.9D 29.7A 8.3A 5.0A 1.9B 2.0A 0.9B 19.4A 1.4B 1.0A tr tr 3.6B 2.5A 6.6B 1.2A 18.4A 1.0A tr 11.0C 42.0B 1.6C nd tr 0.7C nd 1.0C 11.9B 0.6C
6.8 tr 4.1C 14.7B 6.8A 26.8B 4.6B 3.0C 1.2C 1.0C tr 12.1B 1.1C tr tr tr 2.3C 1.3B 3.6C tr 12.2B tr 1.0B 14.4B 33.1C 3.5B 1.4A tr 7.9B 2.8B 12.5B 14.4A 1.2B
tr nd 7.7A 14.8B 2.4C 25.8B 9.5A 4.1B 2.4A 1.4B 1.2AB 19.4A 1.9A 0.7B tr tr 4.0A 2.9A 9.8A 1.1A 20.3A 1.0A 0.6C 9.4D 46.0A tr nd tr 0.8C nd 1.2C 11.6B 0.5C
a
Abbreviations as in Table 1. Data represent the average of three replicate samples. Values for the TL of the six organs with the same letter in one row are not significantly different, values for the PL and TAG fraction of the digestive gland and female gonad with the same capital letter in one row are not significantly different (ANOVA, Tukey HSD, PB0.5). c Total lipid. d Trace (50.5%). b
in female more significantly than in the male gonad. Compared to the male gonad, the proportions of the MUFA, respectively, n-3 PUFA were significantly higher in the female gonad mainly due to higher 16:1n7 and 18:1n-9, 18:3n-3, 18:4n-3 and 20:5n-3 levels, respectively. The fatty acids which distinguished the female gonad from groups I and II were even more abundant in the digestive gland (e.g. the 18:4n-3 and 14:0 levels reached 8.7 and 7.0%, respectively). Apart from the similarities between the digestive gland and the female gonad, the abundance of trimethyltridecanoic acid (TMTD) and 14:0 separated the digestive gland (IV) from the female gonad (III). Separate analysis of the PL and triglycerides (TAG) of the latter organs illustrated that the
high levels of 14:0, 16:1n-7, 18:1n-9, 18:3n-3, 18:4n-3 and EPA in the total lipids of the female gonad and digestive gland originated from the TAG fraction. The TAG of both organs contained more EPA but less DHA than the PL fraction. In both organs, DMA were found nearly exclusively in the PL.
4. Discussion The lower organic matter content of the gills and mantle was in agreement with data for other scallop species such as Chlamys septemradiata [1] and Chlamys islandica [37]. However, there is little information available to compare the results since most data on ash
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Fig. 1. Principal component analysis based on the fatty acids in the total lipids of six organs of the Chilean scallop A. purpuratus. The first and second principal component are plotted in an x– y plane and accounted for 55 and 20%, respectively, of the total sample variance. Group I includes the gills (hollow circle) and mantle (filled circle), group II includes the male gonad (hollow triangle) and the adductor (filled triangle) and group III and IV are formed by the female gonad (hollow rectangular) and digestive gland (filled rectangular), respectively.
content refer to the whole body tissue and are often expressed as % of the total wet weight [5]. The lipid content of the mantle of A. purpuratus (5.3% of the DW) was lower than in the clam Macoma baltica (mantle+siphon, 7.4%) [12] or the mussel Mytilus edulis (9–14%) [46]. Compared to the values reported by Nevenzel et al. [27] for the oyster Crassostrea 6irginica (15.5%) and especially by Klingensmith and Stillway [17] for the hardshell clam Mercenaria mercenaria (26.3–26.5%), quite low lipid concentrations were found in the gills of A. purpuratus (present study) and M. edulis (7–8%) [46]. Values reported in the literature for the lipid content of the gills, expressed as percentage of wet weight, also vary widely, e.g. from 0.6% in the mussel Mytilus californianus and 0.8% in the scallops Placopecten magellanicus and Hinites multirugosus to 1.7% in the mussel Mytilus gallopro6incialis [20,24,27]. In agreement with the fatty acid profile of the gills and mantle of the sea scallop P. magellanicus [25], the EPA levels in the gills and mantle of A. purpuratus were significantly lower than in the other organs. The unidentified fatty acid (22:?) found in A. purpuratus has previously be found in the gonads and larvae of Pecten maximus [23,36] but always at low levels, comparable with the levels found in this study for the gonads, adductor muscle and digestive gland. The very high levels found in the gills and mantle have not been reported earlier and suggest that 22:? has a unique function in these organs although information on its origin and metabolism are completely lacking.
Interestingly, this fatty acid was not present in the gills of the clam M. mercenaria or the scallop P. magellanicus which were both characterized by high levels of 20:1 fatty acid isomers [18,25]. In A. purpuratus, the highest 20:1 levels were detected in the male gonad, followed by the gill, but they were considerably lower than those reported by the latter authors. High 20:1 levels were also reported by Zhukova [47] in the polar lipids of the gills and mantle of M. edulis (12.1 and 10.3%, respectively) together with very high levels of non-methyleneinterrupted dienoic fatty acids (NMID) especially 20:2. Pectinidae normally contains no or only trace levels of NMID [13,14,47]. Although an adequate explanation is not available at present, the results indicate that the 20:1 isomers and NMID are replaced by 22:? in the gills and mantle of A. purpuratus. Other examples of fatty acid profiles of bivalve gills that are remarkably different from that of most other bivalve species can be found in Piretti et al. [30] and Klingensmith [18] for the clams Scapharca inaequi6al6is (12.5% 17:0) and M. mercenaria (26.3 and 44.9% 16:0 and 15.2 and 16.3% 18:0 in the polar and neutral lipids, respectively, and less than 1% DHA in both lipid fractions) and in Fouad et al. [9] for the mussel Mytilus gallopro6incialis (7.6% 18:4n-3, 6.7% 22:1n-9 and 6.3% 16:4). The latter results are distinctly different from the fatty acid profile of other clams and mussels reported in the reviews of Joseph [13,14]. Consistent with available data in literature, the adductor muscle exhibited a low lipid content compared
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Fig. 2. The plot of the factor loadings shows which and how much each fatty acid contributed to the dispersion of the four groups (I, II, III, IV) in Fig. 1. Each fatty acid group (A, B, C, D and E) includes fatty acids which distinguished the individual groups in Fig. 1 (see text for more details). The numbers correspond with the number of each fatty acid and DMA in Table 2.
to the other major organs since glycogen is the main storage form and the fatty acid profile is characterized by a high n-3 PUFA level [1,12,17,24,25]. On the contrary, in M. mercenaria, the lowest n-3 PUFA level was detected in the adductor [18]. In several bivalve species, including A. purpuratus [21], the development of the gonad is associated with a decrease of the carbohydrate reserves in the adductor muscle as glycogen reserves can be used for de novo synthesis of lipids which accumulate in the female gonad during maturation. Similar to other Pectinidae [1,24,39], the lipid level in the male gonad was significantly lower than in the female gonad. Data on the male gonad are scarce compared to the comprehensive information available for the female gonad, and in case of hermaphrodite species, the male and female part are commonly analyzed together [13,22]. The results of studies which allow the fatty acid profiles of the male and female gonad to be compared [25,36] do not yield a uniform trend with respect to the levels of the major fatty acids or fatty acid groups, although 16:0, 20:5n-3 and 22:6n-3 seemed to be always abundant in both organs. The abundance of the group D fatty acids (14:0, 16:1n-7, 18:1n-9, 18:2n-6, 18:3n-3, 18:4n-3) in the TAG fraction of the female gonad and digestive gland clearly distinguished the latter organs from the others (Figs. 1 and 2). The specific anatomical distribution of these fatty acids is associated with the transfer of lipids from the lipid-rich digestive gland to the developing female gonad. This has been observed by several authors who
studied the seasonal lipid or fatty acid composition of both organs [3,25,38,46] and it was also confirmed in 14 C studies with the scallops Chlamys hericia [43] and A. irradiance [2]. On the other hand, Uriarte et al. [41] suggested that the digestive gland is not a storage organ in A. purpuratus since the condition index, defined as (DW organ*100)/(DW total meat), of the digestive gland was very constant during the year whereas the condition index of the adductor and the soft parts (gills+mantle) was negatively correlated with the gonad index. It is well known that during vitellogenesis, large amounts of neutral lipids are accumulating in developing eggs since they provide a major source of energy during embryogenesis [42]. This explains the severe drop of the lipid level in the female gonad of spawned animals [28,40]. TMTD originates from the degradation of dietary chlorophyll. Usually it is only found in substantial amounts in the digestive gland suggesting that it is quickly and efficiently catabolized in the digestive gland before it can be transported to other organs [13,14]. As in the present study, it is sometimes detected at low levels in other organs [13,16,25,30]. In the digestive gland of the scallops P. magellanicus, TMTD was mainly found in the TAG fraction [25]. Due to its characteristic anatomical distribution, the latter authors suggested that it could be a useful indicator of an autotrophic source of reduced carbon and of the nutritional status of the animal through estimation of the TAG reserves. The present study does not support this hypothesis as TMTD was mainly located in the PL and
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not in the TAG fraction of the digestive gland of A. purpuratus. Ether-containing phosphoglycerides are found in nearly all animal cells. In aquatic animals, highest plasmalogen concentrations are found in the gills, nerve and brain tissue. Their functional role in biological systems is not clearly understood, but available information suggest they may play an important function in membrane integrity [7]. Their abundance (detected as DMA) in the gills and mantle of A. purpuratus, two organs which have a great surface in direct contact with the water, appears to add support to this view. Rapport [31] and Nevenzel et al. [27] have previously reported the abundance of plasmalogen in bivalve gill lipids. In contrast with other marine organisms, the DMA composition of bivalves is very poorly documented and none of these studies compared the qualitative or quantitative differences in the DMA profile of different bivalve organs [15,19,32].
Acknowledgements This work has been funded in part by grants from the Flemish government, IWT (Marrit Caers) and the European Union (contract IC18-CT97-0188). We are also grateful to Fondecyt (grant 1970807) and the FONDAP-OMB study group for support as well as the company ‘Salmones Huito’ (Calbuco) for supplying the scallops. We wish to thank P. Soudant, Y. Marty and J.F. Samain for providing well-identified P. maximus FAME extracts.
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