Evolution of fatty acid profiles of subtidal and rocky shore mussel seed (Mytilus galloprovincialis, Lmk.). Influence of environmental parameters

Journal of Experimental Marine Biology and Ecology 268 (2002) 185 – 204 www.elsevier.com/locate/jembe

Evolution of fatty acid profiles of subtidal and rocky shore mussel seed (Mytilus galloprovincialis, Lmk.). Influence of environmental parameters L. Freites, U. Labarta, M.J. Ferna´ndez-Reiriz * CSIC, Instituto de Investigaciones Marinas, C/Eduardo Cabello, 6, 36208 Vigo, Spain Received 28 June 2001; received in revised form 20 September 2001; accepted 12 November 2001

Abstract In the present study fatty acid profiles of Mytilus galloprovincialis mussel seeds originating from two habitats with different environmental conditions (rocky shore and subtidal) were compared after transfer to the same environmental habitat (subtidal). The aim of the research was to investigate the influence of various environmental parameters on the relative percentage of fatty acids. The study was based in the Arosa Rı´a, Northwest Spain, between 27th November 1995 and 3rd July 1996. The location of the mussels suspended from the raft, the rope density (1.6 kg m 1) and cultivation depth (1.5 – 5.0 m) were common parameters for both mussel groups. Our results show that during the first 36 days of the experimental period the mussel origin participated significantly in the model explaining the variance of various fatty acids of physiological importance in marine bivalves, namely the acids 18:0, 16:1n 7, 18:1n 9, 18:1n 7, 18:2n 6, 18:3n 3, 18:4n 3, 20:2NMID1, 20:5n 3 and 22:6n 3. In addition, other environmental parameters related to food availability, such as the ratio chl-a/POM and TPM, only participated in the explanation of two and three of these acids, respectively. In contrast, 50 days into the experiment the mussel origin did not participate in the model of variance of the fatty acids studied, and the ratio chl-a/POM participated significantly in the model explaining the variance of 11 of the total (16) selected fatty acids studied. Moreover, the coefficients were only positive in the fatty acids of known energetic importance in marine bivalves, namely the acids 14:0, 16:1n 7, 18:1n 7, 20:5n 3 and the ratio PUFAs n 3/n 6. The influence that mussel origin and various environmental parameters could exercise on the variability of diverse fatty acids of both mussel groups is discussed. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Different habitats; Fatty acids; Mussel; Mytilus galloprovincialis; Rocky shore and subtidal origin

*

Corresponding author. Tel.: +34-986-231930; fax: +34-986-292762. E-mail address: [email protected] ( M.J. Ferna´ndez-Reiriz).

0022-0981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 9 8 1 ( 0 1 ) 0 0 3 7 7 - X

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1. Introduction Numerous studies have demonstrated that the lipid levels and composition of marine bivalves clearly reflect the biochemical and environmental conditions of seed development (De Moreno et al., 1980; Napolitano et al., 1992; Ferna´ndez-Reiriz et al., 1998; Soudant et al., 1998). From this standpoint, the influence of contrasting or extreme environmental conditions on the biochemical composition of bivalves distributed in such environments would be an interesting study. In this sense, mussels distributed in the rocky shore zone have had to adapt to extreme environments as a consequence of the large tidal ranges. Among these environmental conditions, one of the most important is the reduction in the time available for food acquisition imposed by periods of air exposure (Bayne et al., 1988). With regards to the influence that such periods of starvation usually exercise on lipids, some authors have noted a drop in the triacylglycerols of larval and juvenile stages of different marine bivalves (Fraser, 1989; Caers et al., 2000). The consequence of starvation on different fatty acids in marine bivalves has been highlighted in the work published by Langdon and Waldock (1981), who studied the essential fatty acids PUFAs n 3 and n 6 of oyster juveniles Crassostrea gigas. The authors observed greater decreases in the fatty acid levels of the n 3 family belonging to the triacylglycerol fraction. Bivalves distributed in coastal environments where detritus, bacteria and nanozooplankton contribute substantially to the composition of an abundant food load (Langdon and Newell, 1990) display a relatively high proportion of saturated fatty acids such as 14:0, 16:0 and 18:0 (Perry et al., 1979) compared to bivalves mostly nourished by phytoplankton which are dominated by polyunsaturated fatty acids (Chu et al., 1990; Galap et al., 1999). Various authors have suggested that the seasonal variations observed in the levels of total lipids, neutral lipids and fatty acids of various species of marine invertebrates is intimately related to the nature of their diet. Accordingly, in spring when the available food source is dominated by material of phytoplanktonic origin, an increase in polyunsaturated fatty acids of 18, 20 and 22 carbons has been described (De Moreno et al., 1976b, 1980; Langdon and Waldock, 1981; Ferna´ndez-Reiriz et al., 1996, 1998; Soudant et al., 1999). In the present study, the fatty acid profiles of Mytilus galloprovincialis mussel seeds were analysed over a seasonal cycle in order to ascertain how environmental conditions influence the changes observed in the fatty acid composition. Moreover, comparison of seed from two different habitats (rocky shore and subtidal) will provide information about adaptation delay.

2. Materials and methods 2.1. Specimen acquisition The individuals used in this study were selected from two habitats of contrasting ecological characteristics in the Arosa Rı´a, namely, from the rocky shore zone and from collector ropes suspended from a mussel raft (subtidal environment). Both groups of seeds

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were from the previous spring –summer spawning and therefore belonged to the same annual set. The initial mean size of both groups was 22.55 F 1.55 mm (subtidal) and 19.02 F 1.93 mm (rocky shore), whereas the total dry weight was 0.36 F 0.06 g (subtidal) and 0.27 F 0.06 g (rocky shore), respectively. At the outset, no significant differences in size and initial mass of both mussels groups were noted (ANOVA, P > 0.05). 2.2. Experimental design The experiment was carried out between 27th November 1995 and 3rd July 1996. The mussels from both sets were placed on ropes in the internal zone with a depth of 20 m approximate, near to Arosa Island (Arosa Rı´a, Northwest Spain), and suspended from a classic raft of the type used for the cultivation of this resource. This zone has been characterised as the limit of influence of oceanic waters and is thus affected by rivers and rainfall (winter), and also displays high values of primary productivity (Otto, 1975; Navarro et al., 1991). The location of the mussels on the raft, namely, rope density (1.6 kg m 1) and depth (1.5 –5.0 m) were identical for both seed groups. During the first month the sampling was carried out weekly in order to observe with greater frequency the changes in the biochemical composition of the mussels. Thereafter, sampling was biweekly (January– May) and in the final stage of the experimental period the sampling was monthly (June – July). 2.3. Environmental parameters Seston and corresponding fractions, TPM (Total Particulate Material) = POM (Particulate Organic Material) + PIM (Particulate Inorganic Material), and particulate volume were recorded (six sub-samples) at the same interval depth of the mussel seeds under study. The seston samples were filtered with previously washed Whatman GF/C filters (three times with 10 ml distilled water) and dried (muffle 450
C/4 h). The filtered material was washed with a solution of ammonium formate (0.5 M) to eliminate salts, and dried (110
C/24 h). The organic component (POM) was determined gravimetrically after sample combustion (muffle 450
C/4 h). The particulate volume was determined with a Coulter Counter Multisizer II. The indicators of nutritional quality Q1 (POM/TPM), Q2 (POM/particle vol.1) and the ratio chl-a/POM were subsequently calculated. The data characterising the seston phytoplankton component, expressed as concentration of chl-a, temperature (T ) and salinity were provided by the Marine Environment Quality Control Centre at the Ministry of Fisheries, Shell fisheries and Aquaculture of the Xunta of Galicia (Galician Regional Government). Chl-a was calculated from fluorescence data. 2.4. Sample treatment For each survey three sub-sets of mussels (n = 3), each comprising 30 individuals, were taken at random from both mussel groups, thus making a total of 90 individuals per mussel group. The soft tissues of the individuals of each sub-sample were separated, freeze-dried 1

Particle volume (mm3 l

1

).

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at 70
C under a vacuum of 0.018 mbar and stored at 70
C. Prior to the development of the biochemical analyses the tissues were powdered using a model 6 ‘‘Fritsch’’ pulverisette, and homogenized with water in an ultrasonic vibrator Sonifier 250. 2.5. Analysis of total lipids and fatty acids The lipids were extracted following the method of Bligh and Dyer (1959), modified by Ferna´ndez-Reiriz et al. (1989). The data for the different fatty acids were expressed in relative percentages of the total lipids (%). The fatty acids of total lipids were transesterified to methyl esters with a solution of toluene plus sulphuric acid (H2SO4) in methanol, in a proportion of 1.5:100 ml (Christie, 1982). The esters were then injected into a gas chromatograph (Perkin-Elmer, 8500) equipped with a flame ionisation detector and a 30-m capillary column of flexible silica (Supelco, SP-2330). Nitrogen was used as a carrier gas at a pressure of 10 psi. A programmable (PTV) temperature injector (275
C) was employed, operating in solvent elimination mode (Medina et al., 1994). The temperature of the column was increased from 140 to 210
C at a rate of 1.0
C min 1. The nonadecanoic (19:0) fatty acid was used as an internal standard. Subsequently, relative retention times were calculated and the different fatty acids identified. The results are expressed as relative percentage of each of the different fatty acids identified. 2.6. Statistical analysis With the objective of establishing whether different environmental parameters and the mussel origin factor exercised any influence on the fatty acids values of metabolic importance the experimental period was divided in two sub-periods. The first ran from the start of the experiment up to day 36 and the second from day 50 until completion. Prior to selecting this temporal criterion, the ANOVA results given by Freites et al. (submitted for publication) on the relative percentage of these selected fatty acids of subtidal and rocky shore mussel seed were examined. In this study, Freites et al. (submitted for publication) observed initial differences in 16 fatty acids with physiological importance, and in the case of 16:1n 7, 18:1n 7, 18:3n 3, 18:4n 3 and 20:5n 3 these differences were maintained for longer than two or three week (14 or 21 days). Therefore, the experimental period was divided into two sub-periods with contrasting environmental characteristics, that is to say, one dominated by winter conditions and the other dominated by the spring. Consequently, this permits an adequate evaluation of the influence of environmental parameters to be made over the experimental period. In this way, to study the influence of environmental parameters on the variability of fatty acids of the collector and rocky shore mussel seed, a ‘‘multivariate stepwise regression’’ was performed. In this analysis, the origin factor is attributed to a qualitative factor (dummy) in such a way that the collector mussels are assigned a value of 0 (zero) and those of rocky shore a value of 1 (one). The significance level employed for the regression analysis was 95%. In all cases, the values expressed as relative percentage of the different fatty acids were previously transformed to the arcsine to obtain maximum r2 values (Zar, 1984).

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3. Results 3.1. Environmental parameters With the advance of winter a sustained decrease in water temperature was observed until reaching a minima (12.5
C) (Fig. 1A). Thereafter, a sustained increase of temperature during spring was noted, until peaking in June (16.3
C). Chl-a presented relatively low values during the winter (Fig. 1B), with minimal concentrations (0.61 mg l 1); however, chl-a increases shortly thereafter, reaching maximum values during spring (3.71 mg l 1). Increased differences in the salinity were observed in winter, with low values

Fig. 1. Fluctuations of the environmental parameters temperature (A), chlorophyll-a (B), seston (C), particulate volume (D), food quality (E) and chl-a/POM (F), over the experimental period.

190

Sample Date No. days Fatty acid

1st 27/11/95 0 Subtidals

Rocky shore

Subtidals

Rocky shore

Subtidals

Rocky shore

Subtidals

Rocky shore

Subtidals

Rocky shore

Subtidals

Rocky shore

Subtidals

Rocky shore

14:0 15:0 16:0 17:0 18:0 20:0 22:0 S Sat. 16:1n 16:1n 17:1n 18:1n 18:1n 18:1n 20:1n 20:1n 20:1n 22:1n 22:1n

2.508 0.710 17.938 1.111 6.215 – – 28.482 1.448 5.804 6.333 – 9.119 2.456 1.211 2.235 0.593 – –

1.806 0.749 16.857 2.010 8.154 – – 29.306 1.805 3.703 10.844 – 2.036 1.825 1.790 2.640 0.849 – –

1.758 0.792 16.931 1.727 7.455 – – 28.663 1.915 5.706 8.784 – 4.336 2.405 1.492 2.641 0.884 – –

1.616 0.984 16.465 1.747 7.936 – – 28.747 2.974 3.291 11.271 – 1.864 1.676 1.640 2.870 0.829 – –

0.807 0.395 15.495 1.341 7.759 – – 25.798 1.871 4.358 10.765 – 2.791 1.966 1.654 2.715 0.868 – –

1.732 1.121 16.135 2.247 8.184 – – 29.419 3.037 3.271 12.017 – 1.578 1.627 1.670 2.879 0.889 – –

1.477 0.731 14.850 2.381 7.949 – – 27.388 1.899 3.746 11.388 – 2.151 1.824 1.543 2.681 0.879 – –

1.343 0.912 15.477 2.184 8.333 – – 28.250 2.276 2.928 12.666 – 1.613 1.563 1.471 2.948 0.815 – –

1.190 0.870 14.773 2.074 8.370 – – 27.278 2.202 2.928 12.162 – 2.797 1.748 1.595 3.232 0.912 – –

1.194 1.141 15.101 2.079 9.410 – – 27.574 3.358 2.480 12.055 – 1.461 1.500 1.626 2.843 0.812 – –

1.649 1.193 16.136 2.433 5.882 – – 27.293 3.985 3.293 11.523 – 2.410 1.800 1.363 2.819 0.837 – –

1.370 1.328 15.261 2.343 7.648 – – 27.949 3.951 2.638 10.482 – 1.826 1.823 1.517 2.899 0.874 – –

1.534 1.050 14.847 2.970 7.541 – – 27.943 2.835 3.399 10.188 – 2.007 1.852 1.418 2.784 0.876 – –

1.716 1.031 15.376 3.826 7.402 – – 29.351 3.053 3.914 8.731 – 1.789 2.102 1.534 2.772 0.892 – –

9 7 9 11 9 7 11 9 7 11 9

2nd 05/12/95 8

3rd 13/12/95 15

4th 20/12/95 22

5th 03/01/96 36

6th 17/01/96 50

7th 31/01/96 64

L. Freites et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 185–204

Table 1 Profiles of the different fatty acids of M. galloprovicialis mussel seeds of subtidal and rocky shore origin

29.198 9.067 0.577 0.346 2.268 – 0.673 – 12.931 – 1.620 2.007 – 10.440 0.699 8.857 23.624 – – 2.455 0.717 0.407 2.607 3.172 3.015 42.742 1.826

25.491 1.730 0.969 0.502 3.364 – 0.794 – 7.359 – 1.023 1.367 – 9.012 1.082 12.830 25.314 – – 3.843 1.426 0.550 6.843 5.269 7.394 45.336 3.439

28.162 3.667 0.715 0.390 2.746 – 0.831 – 8.349 – 1.364 1.584 – 11.204 1.539 10.198 25.888 – – 3.250 1.384 0.600 4.003 4.633 4.603 43.473 3.101

26.414 1.616 0.926 0.242 3.571 – 0.891 – 7.247 – 0.978 1.293 – 10.156 0.763 14.134 27.324 – – 3.875 1.341 0.680 4.577 5.216 5.258 45.045 3.770

26.988 1.968 0.941 0.415 3.631 – 0.974 – 7.930 – 0.885 1.212 – 12.316 0.895 12.960 28.268 – – 3.865 1.378 0.690 5.083 5.243 5.773 47.214 3.567

26.967 1.312 1.085 0.592 3.933 – 1.250 – 8.172 – 0.708 0.803 – 9.573 1.369 13.003 25.456 – – 4.248 1.511 0.575 5.053 5.760 5.627 45.015 3.115

26.112 1.574 1.070 0.318 3.885 – 0.765 – 7.612 – 0.870 1.028 – 10.881 2.048 12.283 27.109 – – 4.034 1.688 0.652 5.405 5.722 6.057 46.500 3.569

26.279 1.238 1.082 0.492 4.147 – 1.145 – 8.103 – 0.663 0.794 – 9.506 1.332 12.971 25.266 – – 4.519 1.523 0.636 5.424 6.042 6.060 45.471 3.120

Values are expressed relative percentage of total lipids and represents the mean of three replicates.

27.576 1.866 1.106 0.387 3.902 – 0.603 – 7.865 – 0.645 0.865 – 9.590 1.570 12.132 24.802 – – 4.533 1.880 0.778 5.287 6.413 6.065 45.146 3.158

26.135 1.432 1.105 0.443 4.450 – 0.847 – 8.278 – 0.668 0.981 – 9.776 0.964 14.268 26.657 – – 4.272 1.471 0.645 4.968 5.742 5.613 46.291 3.230

28.030 1.987 1.043 0.526 3.902 – 0.770 – 8.227 – 0.943 1.240 – 10.637 0.829 12.701 26.349 – – 3.725 1.343 0.684 4.349 5.068 5.033 44.677 3.202

26.009 1.819 0.976 0.506 3.769 – 0.689 – 7.760 – 1.021 1.274 – 9.672 0.984 14.807 27.756 – – 3.890 1.339 0.631 4.666 5.229 5.297 46.042 3.578

25.359 1.414 1.036 0.678 3.632 – 1.223 – 7.984 – 0.684 1.036 – 10.752 1.425 13.663 27.560 – – 4.094 1.462 0.612 4.987 5.556 5.599 46.698 3.476

24.787 1.388 0.781 0.631 3.280 – 0.965 – 7.046 – 0.718 1.323 – 11.952 0.949 13.813 28.756 – – 3.689 1.183 0.585 4.604 4.872 5.188 45.862 4.079

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S Monouns. 18:2n 6 18:3n 6 20:3n 6 20:4n 6 22:2n 6 22:4n 6 22:5n 6 Sn 6 16:4n 3 18:3n 3 18:4n 3 20:4n 3 20:5n 3 22:5n 3 22:6n 3 Sn 3 18:3n 4 20:2n 11 20:2NMID1 20:2NMID2 22:2NMID1 22:2NMID2 S 20:NMID S 22:NMID S PUFA n 3/n 6

191

192

Sample Date No. days Fatty acid

8th 15/02/96 80

9th 28/02/96 95

10th 13/03/96 110

11th 27/03/96 125

12th 10/04/96 140

13th 24/04/96 155

14th 05/06/96 197

15th 03/07/96 228

Subtidal Rocky shore

Subtidal Rocky shore

Subtidal Rocky shore

Subtidal Rocky shore

Subtidal Rocky shore

Subtidal Rocky shore

Subtidal Rocky shore

Subtidal Rocky shore

14:0 15:0 16:0 17:0 18:0 20:0 22:0 S Sat. 16:1n 9 16:1n 7 17:1n 9 18:1n 11 18:1n 9 18:1n 7 20:1n 11 20:1n 9 20:1n 7 22:1n 11 22:1n 9 SMonouns. 18:2n 6

2.323 0.710 16.406 1.514 6.959 – – 27.912 1.923 7.881 7.557 – 1.818 2.418 1.278 2.085 1.464 – – 26.422 1.478

2.742 0.927 15.676 2.338 7.309 – – 28.991 2.895 6.068 8.309 – 1.349 2.306 1.177 2.004 1.362 – – 25.470 1.375

2.760 0.802 15.647 1.467 8.015 – – 28.692 2.855 6.447 7.067 – 1.083 2.379 1.173 1.653 1.659 – – 24.315 1.001

3.585 0.519 15.161 0.674 4.074 – – 24.013 1.773 11.783 6.013 – 1.866 2.651 1.002 1.368 1.346 – – 27.804 1.149

3.247 0.517 16.686 0.669 3.915 – – 25.033 1.697 12.610 5.967 – 1.858 2.996 1.111 1.359 1.473 – – 29.071 1.018

3.249 0.381 15.390 0.625 3.749 – – 23.394 0.933 15.847 6.098 – 1.465 3.529 0.868 1.273 1.703 – – 31.717 1.028

4.416 0.416 15.894 0.764 4.273 – – 25.763 1.080 12.041 6.294 – 1.570 3.125 0.843 1.125 1.555 – – 27.632 1.099

2.903 0.427 17.738 0.770 4.533 – – 26.371 1.150 9.895 7.437 – 1.229 3.025 1.200 1.180 1.753 – – 26.870 1.051

1.716 0.966 16.991 1.569 6.395 – – 27.637 2.600 8.310 6.922 – 1.688 2.309 1.092 2.001 1.350 – – 26.272 1.558

2.485 0.763 16.403 1.454 8.059 – – 30.515 2.605 6.043 7.284 – 1.393 2.325 1.303 2.098 1.582 – – 24.633 1.112

2.872 0.660 15.545 0.921 4.776 – – 24.773 2.303 7.566 8.557 – 1.170 2.370 1.341 1.717 1.336 – – 26.361 1.061

3.271 0.482 15.456 0.718 4.219 – – 24.146 1.535 11.133 6.591 – 1.460 2.796 1.042 1.430 1.457 – – 27.443 1.146

4.022 0.468 15.763 0.679 4.219 – – 25.150 1.772 15.109 5.087 – 1.640 3.025 0.828 1.376 1.545 – – 30.381 1.112

3.501 0.410 15.811 0.636 3.870 – – 24.229 1.253 16.930 4.975 – 1.982 3.200 0.816 1.273 1.552 – – 31.982 1.104

4.440 0.377 17.258 0.581 3.869 – – 26.524 0.989 12.139 6.442 – 1.360 3.039 1.026 1.047 1.822 – – 27.864 1.075

2.922 0.416 16.853 0.892 4.974 – – 26.056 0.991 9.450 7.880 – 1.290 2.879 1.047 1.236 1.583 – – 26.357 1.053

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Table 2 Profiles of the different fatty acids of M. galloprovincialis mussel seeds of subtidal and rocky shore origin

0.771 0.430 2.740 – 1.116 – 6.535 – 0.650 2.007 – 17.833 1.465 9.964 31.920 – – 2.452 0.965 0.295 3.499 3.417 3.794 45.666 4.884

0.514 0.593 2.322 – 1.372 – 6.359 – 0.745 2.294 – 18.865 1.578 10.465 33.948 – – 2.268 0.810 0.315 2.389 3.079 2.705 46.090 5.339

0.943 0.652 3.030 – 0.934 – 6.935 – 0.442 1.413 – 16.648 1.479 10.381 30.363 – – 2.534 1.128 0.337 4.242 3.662 4.579 45.539 4.378

0.806 0.578 3.305 – 1.012 – 6.812 – 0.440 1.247 – 16.121 1.365 10.211 29.384 – – 2.520 1.180 0.420 4.537 3.700 4.957 44.852 4.324

0.578 0.461 3.288 – 1.404 – 6.732 – 0.307 1.433 – 21.804 1.704 7.918 33.166 – – 1.997 1.062 0.197 3.839 3.059 4.036 46.993 4.960

0.785 0.610 3.102 – 1.204 – 6.761 – 0.459 1.725 – 23.108 1.618 8.146 35.056 – – 2.159 0.920 0.256 3.713 3.079 3.969 48.866 5.206

0.530 0.560 2.394 – 1.303 – 5.937 – 0.540 1.443 – 28.058 1.399 5.486 36.926 – – 1.652 0.702 0.106 2.860 2.354 2.966 48.183 6.254

0.413 0.604 2.325 – 1.434 – 5.922 – 0.559 1.503 – 27.738 1.478 5.960 37.239 – – 1.624 0.668 0.194 2.764 2.293 2.957 48.411 6.289

0.412 0.644 1.959 – 0.898 – 4.932 – 0.609 1.532 – 26.659 1.543 5.268 35.611 – – 1.571 0.661 0.088 3.034 2.232 3.122 45.896 7.221

Values are expressed as relative percentage of total lipids and represents the mean of three replicates.

0.526 0.561 1.878 – 0.949 – 5.025 – 0.503 1.465 – 26.899 1.262 4.838 34.967 – – 1.404 0.564 0.072 2.435 1.969 2.507 44.469 6.976

0.891 0.534 2.154 – 0.931 – 5.538 – 0.532 1.473 – 25.540 1.360 5.004 33.910 – – 1.427 0.756 0.069 3.189 2.183 3.259 44.889 6.139

0.521 0.601 1.711 – 1.047 – 4.983 – 0.516 1.328 – 26.255 1.465 4.568 34.133 – – 1.324 0.698 0.076 2.575 2.021 2.651 43.789 6.865

0.680 0.607 2.531 – 1.075 – 5.992 – 0.525 1.160 – 26.897 1.349 5.966 35.897 – – 1.081 0.660 0.059 2.915 1.741 2.974 46.604 5.991

0.775 0.594 2.296 – 0.888 – 5.628 – 0.458 1.100 – 27.160 1.358 5.237 35.313 – – 1.083 0.635 0.091 2.862 1.718 2.953 45.612 6.275

0.838 0.548 2.568 – 0.856 – 5.860 – 0.560 1.432 – 26.130 1.567 6.086 35.775 – – 1.228 0.731 0.000 3.165 1.959 3.165 46.759 6.127

0.979 0.506 2.757 – 1.008 – 6.304 – 0.561 1.392 – 24.820 1.690 7.100 35.563 – – 1.336 0.870 0.057 3.458 2.206 3.514 47.587 5.639

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18:3n 6 20:3n 6 20:4n 6 22:2n 6 22:4n 6 22:5n 6 Sn 6 16:4n 3 18:3n 3 18:4n 3 20:4n 3 20:5n 3 22:5n 3 22:6n 3 Sn 3 18:3n 4 20:2n 11 20:2NMID1 20:2NMID2 22:2NMID1 22:2NMID2 S 20:NMID S 22:NMID S PUFA n 3/n 6

193

194

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(22.53) within the upper water layers (0 –5 m) and high values (31.21) at 10– 15 m. Salinity stratification decreased in summer (35.42 at 0– 5 m and 35.74 at 10– 15 m). A series of fluctuations occurred in seston between late November and early February (Fig. 1C). In particular, TPM, POM and PIM were maximum in early January (2.56, 1.00 and 1.29 mg l 1, respectively). Following these augmented concentrations, two new increments in TPM and POM were observed in February (1.34 and 0.57 mg l 1, respectively) and in April (1.381 and 0.64 mg l 1, respectively). Similar events to those described for seston also occur in the particulate volume (Fig. 1D) with an emphatic peak in April (1.66 mm3). Accordingly, three peaks can be observed in the particulate volume, the first cor-

Fig. 2. Variability observed in the relative percentage (% total lipids) of selected fatty acids of the mussel seeds of subtidal (A and C) and rocky shore (B and D) origin, over the experimental period.

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responding with seston maxima at the start of January, and the following two peaks corresponding to those observed in chl-a during spring. With regards to food quality, two clear periods can be discerned in Q2 (Fig. 1E). The first interval between late November and mid February (winter) was characterised with values generally above 0.6, and the second in spring, had values generally below 0.6. These two clearly defined intervals are reflected in the evolution of the ratio chl-a/POM (Fig. 1F). 3.2. Profile of fatty acids identified in the mussels from both origins From a general point of view, the PUFA was the fatty acid group with thePhighest percentage participation in both mussel groups, with values above 42% (see PUFA, Tables 1 and 2). Of these, the total PUFA n 3 fatty acids had a higher range of relative

Table 3 Stepwise multiple regression of saturated (14:0, 16:0, and 18:0), monounsaturated (16:1n 7, 18:1n 9 and 18:1n 7) and PUFA n 6 (18:2n 6 and 20:4n 6) fatty acids of M. galloprovincialis mussel seeds with selected environmental parameters Fatty acids

Parameters

Coefficients

16:0

Constant T r2 = 0.499; Constant TPM Origin r2 = 0.575; Constant Origin TPM T r2 = 0.843; Constant Origin T r2 = 0.546; Constant Origin T r2 = 0.718; Constant Origin T r2 = 0.442; Constant T Origin r2 = 0.572;

12.874 0.716 0.706 F1,28 = 17.933; P < 0.001 16.028 1.475 0.557 2.067 0.514 F2,27 = 11.507; P < 0.001 1.693 2.025 0.633 0.861 0.409 0.766 0.379 F3,26 = 28.743; P < 0.001 11.392 3.931 0.629 1.530 0.387 F2,27 = 10.236; P < 0.001 2.205 0.922 0.695 0.407 0.485 F2,27 = 21.590; P < 0.001 14.974 3.400 0.522 1.696 0.412 F2,27 = 6.740; P < 0.01 21.961 0.776 0.587 0.998 0.477 F2,27 = 11.363; P < 0.001

18:0

16:1n

18:1n

18:1n

18:2n

20:4n

7

9

7

6

6

n = 30,

n = 30,

n = 30,

n = 30,

n = 30,

n = 30,

n = 30,

SE

F-ratio

P

r2

17.933

< 0.001

0.499

12.434 10.581

< 0.01 < 0.01

0.311 0.575

40.989 13.967 11.981

< 0.001 < 0.01 < 0.01

0.401 0.726 0.843

14.846 5.626

< 0.001 < 0.05

0.396 0.546

29.041 14.140

< 0.001 < 0.01

0.483 0.718

8.309 5.171

< 0.01 < 0.05

0.273 0.442

13.685 9.041

< 0.001 < 0.001

0.344 0.572

The various parameters explain the observed variance between the 1st and 5th dates (late autumn – early winter). T = temperature.

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percentages (>23% and < 37%) than the PUFA n 6 (>4% and < 12%), in both mussel groups. With regard to the relative participation of the saturated and monounsaturated fatty acids, both groups presented similar total ranges throughout the study (>23% and < 30%). The NMID fatty acids of 20 and 22 carbons presented relatively low total percentage participation, reaching maximum values below 7%. Regarding the changes over the experimental period, two large groups of fatty acids in both mussel groups can be clearly observed in the relative percentages of the different fatty acids of metabolic importance. The first group comprised the acids presenting lowest values in winter, whereas the highest values were observed in spring. Among the fatty acids which fit this description are 14:0, 16:1n 7, 18:1n 7 and 20:5n 3, along with the sum of the n 3 PUFA and the ratio n 3/n 6, in both mussel groups (Fig. 2A and 2B). In contrast, the second group was comprised of the fatty acids showing highest values in winter and lowest in spring. This group is clearly represented by the fatty acids 18:0, 18:3n 3, 20:4n 6, 22:6n 3, the NMID of 20 and 22 carbons and respective sum, for both rocky shore and subtidal mussels (Fig. 2C and 2D).

Table 4 Stepwise multiple regression of PUFA n 3 (18:3n 3, 18:4n 3, 20:5n 3 and 22:6n 3), NMID (20 and 22 carbons) and the fatty acid ratio n 3/n 6 of M. galloprovincialis mussel seeds with selected environmental parameters Fatty acids

Parameters

18:3n

Constant T Origin r2 = 0.461; n = 30, Constant T Origin r2 = 0.510; n = 30, Constant Origin r2 = 0.987; n = 30, Constant Origin Chl-a/POM r2 = 0.519; n = 30, Constant T Origin r2 = 0.504; n = 30, Constant T r2 = 0.286; n = 30, Constant Chl-a/POM r2 = 0.317; n = 30,

18:4n

20:5n

22:6n

3

3

3

3

20:2NMID1

20:2NMID2

22:2NMID1

Coefficients

SE

3.679 0.639 0.544 0.755 0.406 F2,27 = 7.271; P < 0.01 4.534 0.746 0.599 0.763 0.388 F2,27 = 8.832; P < 0.01 19.218 1.270 0.647 F1,28 = 12.981; P < 0.01 20.952 1.919 0.614 1.063 0.377 F2,27 = 9.163; P < 0.01 21.155 0.686 0.564 0.830 0.431 F2,27 = 8.643; P < 0.01 14.842 0.538 0.535 F1,28 = 7.214; P < 0.05 5.084 0.432 0.563 F1,28 = 8.365; P < 0.01

P

r2

9.338 5.203

< 0.01 < 0.05

0.296 0.461

12.452 5.211

< 0.01 < 0.05

0.359 0.510

12.981

< 0.01

0.987

13.300 5.025

< 0.01 < 0.05

0.377 0.519

10.906 6.379

< 0.01 < 0.05

0.318 0.504

7.214

< 0.001

0.286

8.365

< 0.01

0.317

F-ratio

The various parameters explain the observed variance between the 1st and 5th dates (late autumn – early winter).

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3.3. Influence of mussel origin and environmental parameters on the variability of the different fatty acids 3.3.1. Mussel origin In general, the stepwise multiple regression analysis showed that during the first 36 days of the experiment (end of autumn– start of winter), the mussel origin participated significantly ( P < 0.05) in the variance observed in 11 (69%) of the 16 fatty acids considered to be of metabolic importance (Tables 3 and 4). These fatty acids were the following: 18:0, 18:2n 6, 20:4n 6, 18:3n 3, 18:4n 3, 20:5n 3, 22:6n 3 and 20:2NMID1. The coefficient of the origin term is negative for those fatty acids with known energetic importance, such as 16:1n 7, 18:1n 9, 18:1n 7, 18:2n 6 and 20:5n 3, thus demonstrating the higher relative percentage (% total lipids) in subtidal mussels (qualitative value 0) than rocky shore mussels (qualitative value 1). Therefore, where the mussel origin participated in the explanation of variance, the explanation percentages were elevated to values at least 26% higher (18:0, 16:1n 7, 18:1n 9, 18:1n 7, 18:2n 6,

Table 5 Stepwise multiple regression of saturated (14:0, 16:0, and 18:0), monounsaturated (16:1n 7, 18:1n 9 and 18:1n 7) and PUFA n 6 (18:2n 6 and 20:4n 6) of M. galloprovincialis mussel seeds with selected environmental parameters Fatty acids

Parameters

14:0

Constant Chl-a/POM T r2 = 0.478; n = 60, Constant T r2 = 0.585; n = 60, Constant Chl-a/POM T TPM r2 = 0.810; n = 60, Constant Chl-a/POM T r2 = 0.708; n = 60, Constant Chl-a/POM r2 = 0.310; n = 60, Constant Chl-a/POM TPM r2 = 0.375; n = 60,

18:0

16:1n

18:1n

18:2n

20:4n

7

7

6

6

Coefficients

SE

2.877 0.807 0.475 0.729 0.309 F2,57 = 16.942; P < 0.001 46.258 2.338 0.765 F1,58 = 53.465; P < 0.001 35.647 2.373 0.527 3.082 0.493 3.308 0.220 F3,56 = 51.260; P < 0.001 0.511 0.454 0.490 0.607 0.472 F2,57 = 44.803; P < 0.001 7.494 0.398 0.557 F1,58 = 17.083; P < 0.001 14.401 2.663 0.334 1.164 0.487 F2,57 = 11.110; P < 0.001

F-ratio

P

r2

11.491 4.862

< 0.01 < 0.05

0.409 0.478

53.465

< 0.001

0.585

35.167 28.938 8.014

< 0.001 < 0.001 < 0.01

0.653 0.768 0.810

21.774 20.194

< 0.001 < 0.001

0.548 0.708

17.083

< 0.001

0.310

13.922 6.555

< 0.001 < 0.05

0.265 0.375

The various parameters explain the observed variance between the 6th and 15th dates (winter – early summer). T = temperature.

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20:4n 6, 20:5n 3 and 22:6n 3). Of these it is important to highlight the high percentage owing to the origin term (98.7%) on the explanation of the variance observed in the fatty acid 20:5n 3. In the remaining fatty acids the origin term raises the explanation percentage to at least 15– 23% in the majority of cases. From day 50 until the end of the experiment the mussel origin did not participate in the variance of any of the fatty acids of known metabolic importance (Tables 5 and 6).

Table 6 Stepwise multiple regression of PUFA n 3 (18:3n 3, 18:4n 3, 20:5n 3 and 22:6n 3), NMID (20 and 22 carbons) and the fatty acid ratio n 3/n 6 of M. galloprovincialis mussel seeds with selected environmental parameters Fatty acids

Parameters

18:3n

Constant Chl-a/POM T r2 = 0.315; n = 60, Constant TPM r2 = 0.333; n = 60, Constant Chl-a/POM T r2 = 0.572; n = 60, Constant Chl-a/POM T r2 = 0.604; n = 60, Constant Chl-a/POM T r2 = 0.614; n = 60, Constant Chl-a/POM T TPM r2 = 0.669; n = 60, Constant T Chl-a/POM r2 = 0.462; n = 60, Constant Chl-a/POM T TPM r2 = 0.681; n = 60,

18:4n

20:5n

22:6n

3

3

3

3

20:2NMID1

20:2NMID2

22:2NMID1

n

3/n

6

Coefficients

SE

0.456 4.388 0.661 0.367 0.398 F2,57 = 8.521; P < 0.001 5.584 1.372 0.577 F1,58 = 19.005; P < 0.001 5.245 2.838 0.586 1.749 0.260 F2,57 = 24.748; P < 0.001 47.837 1.673 0.487 1.907 0.400 F2,57 = 28.206; P < 0.001 25.781 0.899 0.480 1.076 0.414 F2,57 = 29.416; P < 0.001 15.022 0.378 0.444 0.565 0.477 0.694 0.244 F3,56 = 24.240; P < 0.001 18.419 1.040 0.454 0.526 0.319 F2,57 = 15.911; P < 0.001 3.861 0.784 0.485 0.979 0.436 1.396 0.259 F3,56 = 25.669; P < 0.001

F-ratio

P

r2

16.927 6.130

< 0.001 < 0.05

0.202 0.315

19.005

< 0.001

0.333

21.278 4.186

< 0.001 < 0.05

0.524 0.572

15.896 10.697

< 0.001 < 0.01

0.489 0.604

15.869 11.785

< 0.001 < 0.001

0.491 0.614

14.276 15.548 5.642

< 0.001 < 0.001 < 0.05

0.516 0.617 0.669

10.191 5.024

< 0.01 < 0.05

0.389 0.462

17.725 13.496 6.593

< 0.001 < 0.001 < 0.05

0.546 0.623 0.681

The various parameters explain the observed variance between the 6th and 15th dates (winter – early summer). T = temperature.

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3.3.2. Availability of TPM In general, over the first 36 days, the TPM term only participated significantly ( P < 0.01) with highest percentage explanation of variance (>30%) in two fatty acids (18:0 and 16:1n 7) with metabolic importance (Tables 3 and 4). From day 50 until the end of the experiment TPM gained superiority in the explanation of variance with regards to fatty acids of metabolic importance. During this period, TPM participated in the model which significantly explains ( P < 0.05) the variance of the 16:1n 7, 20:4n 6, 18:4n 3, 20:2NMID2 fatty acids and in ratio PUFAs n 3/n 6 (Tables 5 and 6). In the case of 20:4n 6 and 20:2NMID2 fatty acids, the coefficient is negative. 3.3.3. Ratio chlorophyll-a/POM During the first 36 days the chl-a/POM ratio only participated significantly ( P < 0.001) in the model explanation of fatty acids for 22:6n 3 and 22:2NMID1 (Tables 3 and 4). There was an inverse relationship in these fatty acids with known structural functions. In contrast, after 50 days the ratio chl-a/POM participates highly significantly ( P < 0.001) with high percentages (>40%) of explanation of the variance (Tables 5 and 6), particularly with the fatty acids characterised by a mainly energetic function, such as the 14:0, 16:1n 7, 18:1n 7, 18:2n 6, 20:5n 3 and the ratio PUFA n 3/n 6. Chla/POM showed a positive relationship with these fatty acids and with the PUFA n 3/ n 6 relationship, which highlights the direct relationship with these fatty acids. This relationship was inverse (Tables 5 and 6) in the fatty acids with known structural functions (18:0, 18:3n 3, 22:6n 3 and NMID of 20 carbons) or those related to reproduction (20:4n 6). 3.3.4. Temperature In the first 36 days, the temperature participated significantly ( P < 0.05) in the model explains of the variance observed in 10 of the selected fatty acid with physiological importance. Of these, temperature had an inverse relationship with the fatty acids related to reproduction, such as 20:4n 6. After 50 days the water temperature participated significantly ( P < 0.001) in explaining the variance of the majority of fatty acids (Tables 5 and 6). Exceptions include the fatty acid 18:2n 6, 20:4n 6 and 18:4n 3. Furthermore, for the acids required as energy, as such 16:1n 7, 18:1n 7 and 20:5n 3, this environmental parameter gave a positive relationship (Tables 5 and 6).

4. Discussion The stepwise multiple regression analysis showed that during the first period up to day 36 the origin term of the seeds participated in the explanation of the variance for 11 (69%) of the fatty acids with recognised metabolic importance in marine bivalves. Of these fatty acids, the origin participates on four of those fatty acids responsible for energetic functions, such as the 20:5n 3, among others (Gardner and Riley, 1972). In these cases, the coefficient of the mussel origin term was always negative, thus indicating a lower

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content of these fatty acids in the rocky shore mussels. This lower initial content could be a consequence of the feeding times imposed on the mussels in the coastal strip by periods of air exposure. In this sense, it has been shown that such periods have a similar effect to starvation (Hummel et al., 1989). Correspondingly, Langdon and Waldock (1981) showed that juveniles of the oyster C. gigas subjected to starvation showed differential decreases in the n 3 family of fatty acids belonging to the triacylglycerols. However, the association of the influence of the origin term and the qualitative and quantitative differences of the food present in the rocky shore and subtidal habitats should not be ruled out. Fluctuations in the concentration and quality of seston in the rocky shore environment are frequent, whereas in the cultivation area these parameters are relatively stable (Labarta et al., 1997). In bivalves distributed in environments characterised by a ready available supply of non-phytoplanktonic organic material and with an abundant bacterial load, a high proportion of saturated fatty acids such as the 20:0 has been observed (Galap et al., 1999). Marine phytoplankton are dominated by the polyunsaturated fatty acids n 3 of 20 and 22 carbons, and this composition is reflected in bivalves mainly nourished by this food source (Webb and Chu, 1983; Langdon and Waldock, 1981; Bell and Sargent, 1985; Besnard et al., 1989; Chu et al, 1990). This could explain, for example, the higher content of the fatty acid 18:0 in mussels of rocky shore origin, since marine detritus contains substantial quantities of saturated fatty acids between 14 and 18 carbons (Ackman et al., 1968; Chuecas and Riley, 1969; Perry et al., 1979). Aside from mussel origin, the multivariate regression analysis showed that during the first 36 days the terms related to food availability only participated in the explanation of the variability of two fatty acid levels, such as 18:0 and 16:1n 7 (TPM) and 22:6n 3 and 22:2NMID1 (ratio chl-a/POM). This is probably due in part to the relatively low availability of phytoplanktonic food and the erratic evolution of the TPM observed during this period. The ratio chl-a/POM showed a direct relationship with the content of fatty acids of mainly energetic function (e.g. 16:0, 18:1n 9, 18:1n 7, 18:2n 6 and 20:5n 3). The highly significant positive relationship of 16:1n 7, 18:1n 7 and 20:5n 3 fatty acids with chl-a/POM suggests that these acids are directly derived from the phytoplanktonic diet, given the previous considerations that marine phytoplankton is the greatest source of 18:1n 7 and 18:1n 9 fatty acids (Delaunay et al., 1993; Napolitano et al., 1992) among others. From day 50 to the end of the experiment the seed origin did not participate significantly in the explanation of variance of any of the fatty acids with metabolic importance. This demonstrates that the rocky shore seeds accomplish complete metabolic adaptation from the start of the experimental to day 36. In contrast to the first 36 days, the chl-a/POM ratio develops greater precedence as the term explaining the variance observed in the fatty acids. Accordingly, chl-a/POM participates positively and significantly in explaining the variance of the almost all the fatty acids of metabolic importance studied, in particular the fatty acids recognised as being mainly energetic in character, such as 14:0, 16:1n 7, 18:1n 9, 18:1n 7, 18:2n 6, 20:5n 3 and also the ratio PUFAs n 3/n 6. The highest percentages with respect to explanation (>52%) of variability were recorded for the 16:1n 7, 18:1n 7 and 20:5n 3 fatty acids, coinciding with the period of greater availability of phytoplanktonic food (spring – summer, see Fig. 1B). Phytoplankton is the greatest food source for

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bivalve molluscs and is considered the principal source in marines trophic chains for linoleic (18:2n 6), linolenic (18:3n 3) and eicosapentaenoic (20:5n 3) polyunsaturated fatty acid (Sargent, 1976). Similarly, some species of diatoms are especially rich in the fatty acids 18:1n 9, 18:4n 3 and 20:5n 3 (Ackman et al., 1968; Chuecas and Riley, 1969; Ferna´ndez-Reiriz et al., 1989; Napolitano et al., 1992). When phytoplanktonic food is readily available, these results agree with previous findings of increases in the fatty acid levels of the bivalves Mesodesma mactroides (De Moreno et al., 1976a), M. edulis (Waldock and Holland, 1979) and M. galloprovincialis (Ferna´ndez-Reiriz et al., 1996). Furthermore, high levels of these fatty acids have been observed in larval and post-larval stages of various marine bivalve species nourished by microalgal diets characteristically rich in these fatty acids (Langdon and Waldock, 1981; Albentosa et al., 1994, 1999; Ferna´ndez-Reiriz et al., 1998, 1999; Soudant et al., 1998; Labarta et al., 1999; Caers et al., 2000). Of the total acids studied in both mussel groups, the fatty acid group with the greatest percentage participation were the polyunsaturated fatty acids (PUFAs). These results agree with published findings regarding other marine bivalves where the fatty acid group of greatest participation from the total were the PUFAs (Bell and Sargent, 1985; Besnard et al., 1989; Pazos et al., 1996, 1997). Furthermore, within the polyunsaturated group the n 3 series and, more specifically, the 20:5n 3 and 22:6n 3 fatty acids, were those contributing the highest percentages. These results agree with those obtained by Abad et al. (1995) and Labarta et al. (1999) who observed that the PUFAs and sub-series PUFAs n 3 showed the greatest values for the fatty acid profiles of the adult and larvaepostlarvae (respectively) of the oyster Ostrea edulis. An interesting feature within the PUFAs n 6 group is the significantly higher values presented by the fatty acid 20:4n 6 in both mussel groups during January (ANOVA, Tukey P < 0.001) when minimum phytoplanktonic food concentrations and POM (winter) were registered. These levels probably arise from selective retention of the 20:4n 6 fatty acid for use in reproductive processes (Osada et al., 1989) or in the incorporation of the structural lipids of the eggs (Soudant et al., 1996), rather than from incorporation by food ingestion, since coinciding with this period the mussels of both origins are habitually found in a state of gametogenesis, as shown by Ferran et al. (1990) and Villalba (1995). Accordingly, it has been shown that the acid 20:4n 6 in the oyster C. virginica can be implicated in the synthesis of various neurotransmitters related to reproductive processes, such as the prostaglandins 2 (Nomura et al., 1979). These findings coincide with the results reported by Pazos et al. (1996) in specimens of the oyster C. gigas cultivated from the same rı´a and, moreover, in which highest levels of this fatty acid were observed in January.

Acknowledgements The authors would like to thank Dr. J. L. Garrido, Dr. J.F. Babarro, Ana Ayala, Beatriz Gonza´lez, Lourdes Nieto and Sonia Villar for their technical assistance in the biochemical analyses, and the crew of the ‘‘Jose´ Marı´a Navaz’’ from the Instituto Espan˜ol de Oceanografı´a. This study was financed by the project CICYT MAR97-0592. Luis Freites

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works in the Instituto Oceanogra´fico de Venezuela, Universidad de Oriente, and his research was supported by a grant from the Consejo Nacional de Investigaciones Cientı´ficas y Tecnolo´gicas (CONICIT), Venezuela. Finally, we would like to thank two anonymous reviewers whose comments improved this manuscript. [SS]

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