Reduced dietary levels of EPA and DHA have a major impact on the composition of

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1 Supplementary materials Reduced dietary levels of EA and DHA have a major impact on the composition of skin membrane lipids in Atlantic salmon (Salmo salar L.) Ken Cheng *,, Marta Bou, Bente Ruyter, Jana ickova, Emad Ehtesham, Liang Du, Claudia Venegas, Ali A. Moazzami Department of Molecular Sciences, Uppsala BioCenter, Swedish University of Agricultural Sciences,.O. Box 7015, Uppsala, Sweden Norwegian Institute of Food, Fisheries and Aquaculture Research (Nofima), Box 210, NO Ås, Norway AVS Chile S.A., Casilla 300, uerto Varas, Chile *Tel.: ; address: cheng-ken@live.cn

2 C Score plot Loading plot a b E c d S e f I g h Figure S1. rincipal component analysis (CA) score plots and loading plots created with the fatty acid profile data in different glycerol-phospholipid fractions in skin samples of 200 g fish fed different diets for 19 weeks. C, phosphatidylcholine: E, phosphatidylethanolamine: S, phosphatidylserine: I, phosphatidylinositol. (a) CA score plot and (b) loading plot for the C fraction. The CA model was established using seven principal components (R 2 X=94.8%, Q 2 =59.5%). The first (C1) and the second principal component (C2) explained 43.5 and 21.5% of the data variance, respectively. (c) CA score plot, and (d) loading plot for the E fraction. The CA model was established using three principal components (R 2 X=83.1%, Q 2 =60.5%). C1 and C2 explained 43.7 and 24.9% of the data variance, respectively. (e) CA score plot and (f) loading plot for the S fraction. The CA model

3 was established using five principal components (R 2 X=90.6%, Q 2 =58.7%). C1 and C2 explained 56.4 and 14.8% of the data variation, respectively. (g) CA score plot and (h) loading plot for the I fraction. The CA model was established using two principal components (R 2 X=70.2%, Q 2 =47.4%), with 47.4 and 22.8% of the data variation explained by C1 and C2, respectively.

4 Table S1. Chemical composition (% of total feed content) and gross energy (MJ.kg -1 ) of the experimental diets. Chemical composition CC 0% EA+DHA 0.5% 1.0% 1.5% 2.0% 0.5% 1.0% 1.5% 2.0% 0.5% 1.0% 1.5% 2.0% Dry matter (%) roteins (%) Fat (%) Ash (%) Gross energy (MJ Kg- 1 ) Table S2. Absolute concentrations of sphingolipids (nmol/g) and calculated ratio of sphingomyelin to ceramide (Sph/Cer) and glucosylceramide to ceramide (GlcCer/Cer) in skin of Atlantic salmon fed the commercial-type control (CC) and experimental diets for 19 weeks (200 g) and 26 weeks (400 g; mean±se; n=2 for 0.5% groups, n=3 for other groups 1 ). Sphingolipids Time 0% EA+DHA 0.5% groups 2.0% groups CC -Diet -Time C14:0 Cer 200 g 3.14± ± ± ± ± ± ± ±0.13 < < g 1.93± ± ± ± ± ± ± ±0.28 C16:0 Cer 200 g 1.63± ± ± ± ± ± ± ± g 1.49± ± ± ± ± ± ± ±0.14 C18:0 Cer 200 g 0.39± ± ± ± ± ± ± ± g 0.38± ± ± ± ± ± ± ±0.01 C24:1 Cer 200 g 3.50± ± ± ± ± ± ± ±0.19 < < g 2.48± ± ± ± ± ± ± ±0.18 C24:2 Cer 200 g 1.23± ± ± ± ± ± ± ± < g 0.85± ± ± ± ± ± ± ±0.06 Cer 200 g 9.89± ± ± ± ± ± ± ± < g 7.13± ± ± ± ± ± ± ±0.66 d18:1 So 200 g 1.73± ± ± ± ± ± ± ± g 2.05± ± ± ± ± ± ± ±0.03 d20:1 So 200 g 0.09± ± ± ± ± ± ± ± < g 0.08± ± ± ± ± ± ± ±0.01 So 200 g 1.82± ± ± ± ± ± ± ± g 2.13± ± ± ± ± ± ± ±0.03 d18:0 Sa 200 g 0.15±0.01 ab 0.18±0.07 ab 0.11±0.01 b 0.23±0.03 a 0.19±0.05 ab 0.14±0.01 ab 0.25±0.01 a 0.06± g 0.12±0.03 ab 0.10±0.01 ab 0.09±0.00 b 0.15±0.03 a 0.14±0.02 ab 0.10±0.01 ab 0.14±0.01 a 0.05±0.01 d20:0 Sa 200 g 0.04±0.00 ab 0.03±0.00 b 0.03±0.00 b 0.05±0.01 ab 0.04±0.01 ab 0.05±0.01 ab 0.06±0.00 a 0.02± g 0.03±0.01 ab 0.04±0.00 b 0.03±0.00 b 0.04±0.01 ab 0.04±0.01 ab 0.04±0.01 ab 0.05±0.00 a 0.02±0.00

5 Sa 200 g 0.20±0.01 ab 0.21±0.07 ab 0.14±0.02 b 0.29±0.04 aa 0.23±0.06 ab 0.19±0.02 ab 0.31±0.01 a 0.07± g 0.15±0.03 ab 0.14±0.01 ab 0.12±0.01 b 0.19±0.04 ab 0.19±0.03 ab 0.14±0.02 ab 0.19±0.01 a 0.07±0.01 C14:0 GlcCer 200 g 3.53±0.36 a 3.17±0.45 abc 3.52±0.02 ab 3.01±0.07 abc 2.97±0.13 abc 2.46±0.10 c 2.53±0.11 bc 3.77±0.27 < < g 2.67±0.17 a 1.87±0.01 abc 1.97±0.11 ab 2.16±0.06 abc 2.20±0.20 abc 1.55±0.20 c 1.96±0.07 bc 3.43±0.04 C16:0 GlcCer 200 g 3.83±0.49 a 3.40±0.01 ab 3.52±0.12 ab 3.21±0.24 ab 3.28±0.15 ab 2.99±0.17 b 2.74±0.01 b 2.14± g 3.67±0.25 a 2.79±0.04 ab 3.27±0.56 ab 3.26±0.08 ab 2.82±0.04 ab 2.70±0.14 b 2.78±0.17 b 2.52±0.13 C18:0 GlcCer 200 g 0.20± ± ± ± ± ± ± ± g 0.21± ± ± ± ± ± ± ±0.02 C24:1 GlcCer 200 g 3.35± ± ± ± ± ± ± ± g 2.81± ± ± ± ± ± ± ±0.10 C24:2 GlcCer 200 g 1.29±0.07 a 1.24±0.01 ab 1.21±0.02 a 1.20±0.08 a 1.28±0.08 a 0.96±0.06 b 1.16±0.05 a 0.76± g 1.24±0.09 a 1.03±0.06 ab 1.34±0.06 a 1.23±0.10 a 1.32±0.03 a 0.92±0.04 b 1.18±0.09 a 0.69±0.02 GlcCer 200 g 12.19±1.20 a 11.2±0.51 abc 11.8±0.36 ab 10.4±0.39 abc 10.7±0.46 abc 9.38±0.38 c 9.33±0.21 bc 9.03±0.54 < < g 10.61±0.62 a 8.32±0.02 abc 9.49±0.80 ab 9.32±0.11 abc 9.44±0.49 abc 7.57±0.64 c 8.28±0.38 bc 8.99±0.25 C14:0 Sph 200 g 123.6± ± ± ± ± ± ± ±5.94 < < g 95.47± ± ± ± ± ± ± ±7.13 C16:0 Sph 200 g 68.74±4.73 a 56.86±2.97 ab 56.92±0.73 ab 56.67±10.7 ab 60.62±3.47 b 59.07±4.28 b 52.94±3.69 ab 47.23± g 75.13±3.40 a 59.29±5.84 ab 59.04±5.80 ab 66.49±2.20 ab 53.00±0.70 b 54.91±3.71 b 63.75±2.23 ab 49.38±1.27 C18:0 Sph 200 g 10.75±1.35 a 7.65±0.10 ab 7.34±0.14 b 8.47±1.25 ab 8.50±0.34 ab 7.41±0.66 ab 8.37±1.84 ab 3.20± g 10.83±0.59 a 9.41±0.84 ab 8.44±0.19 b 11.0±0.22 ab 7.73±0.49 ab 8.55±0.93 ab 10.2±0.90 ab 3.06±0.06 C20:2 Sph 200 g 0.75± ± ± ± ± ± ± ± g 0.63± ± ± ± ± ± ± ±0.03 C22:0 Sph 200 g 5.10±0.27 a 4.64±0.38 ab 4.39±0.32 ab 4.48±0.28 ab 4.65±0.24 ab 3.84±0.04 b 3.97±0.62 b 3.12± g 4.86±0.31 a 3.99±0.30 ab 3.47±0.00 ab 3.89±0.07 ab 3.47±0.35 ab 3.45±0.55 b 3.65±0.11 b 2.89±0.05 C22:1 Sph 200 g 7.36± ± ± ± ± ± ± ±0.43 < < g 5.68± ± ± ± ± ± ± ±0.17 C24:1 Sph 200 g 118.4± ± ± ± ± ± ± ± < g 98.72± ± ± ± ± ± ± ±1.78 C24:2 Sph 200 g 32.52±1.05 b 35.04±3.56 ab 35.45±1.97 ab 30.61±4.61 b 45.27±2.42 a 38.51±2.47 ab 34.07±3.85 ab 25.41± g 30.80±1.63 b 34.16±2.10 ab 31.73±2.84 ab 32.44±0.06 b 37.11±4.67 a 34.04±3.59 ab 34.92±1.44 ab 18.29±0.51 Sph 200 g 367.2± ± ± ± ± ± ± ± < g 322.1± ± ± ± ± ± ± ±10.5 C14:0 Sph/Cer 200 g 39.38± ± ± ± ± ± ± ± g 49.84± ± ± ± ± ± ± ±1.69 C16:0 Sph/Cer 200 g 42.84±2.09 a 39.39±0.78 ab 37.06±0.46 ab 38.13±0.72 ab 37.21±3.22 b 38.61±5.27 ab 34.63±5.09 ab 35.81± g 50.51±1.06 a 39.44±8.57 ab 34.80±2.82 ab 46.18±5.10 ab 30.16±2.71 b 35.31±2.96 ab 45.46±2.44 ab 45.79±4.02 C18:0 Sph/Cer 200 g 27.24± ± ± ± ± ± ± ± g 28.87± ± ± ± ± ± ± ±3.11 C24:1 Sph/Cer 200 g 34.20± ± ± ± ± ± ± ± g 39.97± ± ± ± ± ± ± ±3.41 C24:2 Sph/Cer 200 g 27.59± ± ± ± ± ± ± ± g 36.35± ± ± ± ± ± ± ±4.65

6 Sph/Cer 200 g 37.45± ± ± ± ± ± ± ± g 45.30± ± ± ± ± ± ± ±2.76 C14:0 GlcCer/Cer 200 g 1.12±0.07 a 1.13±0.00 ab 1.10±0.04 ab 1.02±0.18 ab 1.01±0.09 ab 0.87±0.12 b 0.84±0.05 ab 0.95± g 1.40±0.13 a 1.07±0.10 ab 0.94±0.09 ab 1.11±0.13 ab 1.06±0.09 ab 0.81±0.10 b 1.11±0.01 ab 1.26±0.11 C16:0 GlcCer/Cer 200 g 2.37±0.12 a 2.36±0.07 ab 2.29±0.13 ab 2.18±0.19 ab 2.01±0.12 b 1.93±0.08 b 1.79±0.14 b 1.64± g 2.46±0.04 a 1.83±0.20 ab 1.94±0.36 ab 2.28±0.38 ab 1.60±0.12 b 1.73±0.12 b 1.96±0.08 b 2.36±0.35 C18:0 GlcCer/Cer 200 g 0.50± ± ± ± ± ± ± ± g 0.57± ± ± ± ± ± ± ±0.11 C24:1 GlcCer/Cer 200 g 0.96± ± ± ± ± ± ± ± g 1.13± ± ± ± ± ± ± ±0.11 C24:2 GlcCer/Cer 200 g 1.08±0.11 a 1.17±0.03 ab 1.13±0.06 ab 1.20±0.05 a 1.07±0.06 ab 0.87±0.06 b 1.01±0.06 ab 1.01± < g 1.47±0.17 a 1.20±0.11 ab 1.36±0.08 ab 1.39±0.04 a 1.21±0.08 ab 1.08±0.06 b 1.37±0.06 ab 1.57±0.18 GlcCer/Cer 200 g 1.23±0.02 a 1.27±0.02 ab 1.26±0.07 ab 1.21±0.12 ab 1.15±0.05 ab 1.04±0.07 b 1.00±0.05 ab 1.00± g 1.49±0.07 a 1.20±0.08 ab 1.22±0.11 ab 1.33±0.14 ab 1.20±0.10 ab 1.08±0.10 b 1.23±0.02 ab 1.39±0.14 Cer ceramide; GlcCer glucosyl-ceramide; Sa sphinganine; So sphingosine; Sph sphingomyelin. 1 Each statistical replicate originated from a pooled sample of skin from five fish, measured three times. values calculated by two-way ANOVA or Mann-Whitney test (Tukey s test) within the dietary groups except CC, using diet and sampling time as fixed factors. ab Different superscript letters indicate significant differences between diets from both time points (Tukey s test. <0.05). Table S3a. Fatty acid (FA) composition (% of total identified) in the phosphatidylcholine (C) fraction in skin of fish at 400 g weight (mean±se; n=3 for commercial-type control (CC), 0% EA+DHA, and 2% EA and/or DHA, n=2 for other groups 1 ). FA CC 0% EA+DHA 0.5% 1.0% 1.5% 2.0% 0.5% 1.0% 1.5% 2.0% 0.5% 1.0% 1.5% 2.0% 12:0 0.39± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 1.51± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 24.2± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 3.12± ±0.11 a 4.22±0.12 ab 3.80±0.15 ab 3.86±0.08 ab 4.15±0.06 ab 3.90±0.18 ab 3.77±0.12 ab 3.66±0.01 b 3.76±0.17 ab 4.30±0.13 a 3.72±0.02 ab 3.76±0.06 ab 3.65±0.05 b :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ±0.17 a 21.3±0.27 ab 21.5±1.21 ab 20.0±1.48 abc 20.1±0.45 abc 21.0±0.27 abc 20.4±0.33 abc 20.5±0.28 abc 18.1±0.34 c 20.4±0.08 abc 21.0±0.64 abc 19.6±0.02 abc 19.5±0.30 bc :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ±0.26 a 8.34±0.37 b 6.55±0.11 de 5.63±0.06 efg 4.70±0.09 g 8.37±0.57 b 8.07±0.45 bc 6.77±0.10 cde 5.94±0.18 ef 8.33±0.29 b 7.26±0.10 bcd 5.89±0.11 ef 5.07±0.12 fg < :3n ± ±0.07 a 0.50±0.03 b 0.34±0.00 bcd 0.22±0.03 cd 0.17±0.01 d 0.43±0.03 bc 0.39±0.07 bcd 0.22±0.02 cd 0.17±0.01 d 0.46±0.04 bc 0.49±0.21 bc 0.23±0.01 bcd 0.16±0.02 d < :2n ± ±0.02 c 0.95±0.02a bc 0.79±0.04 abc 0.84±0.01 abc 0.71±0.03 bc 0.75±0.32 abc 1.02±0.05 abc 1.08±0.04 ab 1.03±0.03 abc 1.03±0.11 abc 1.14±0.03 a 0.91±0.04 abc 0.80±0.09 abc :3n ± ±0.09 a 3.35±0.11 b 2.05±0.07 d 1.42±0.01 e 1.00±0.04 e 3.04±0.14 bc 2.52±0.44 cd 1.35±0.03 e 1.01±0.03 e 2.75±0.27 bcd 2.10±0.00 d 1.43±0.04 e 0.94±0.02 e < :4n ± ±0.17 a 6.41±0.15 bc 4.73±0.00 de 3.66±0.08 ef 3.24±0.04 f 7.39±0.45 b 5.68±0.43 cd 4.44±0.30 de 4.37±0.15 ef 6.23±0.76 bc 4.61±0.03 de 4.10±0.08 ef 3.67±0.11 ef <0.0001

7 22:5n ± ±0.04 ab 0.63±0.10 bcde 0.36±0.00 de 0.42±0.10 cde 0.24±0.04 e 0.86±0.53 abcd 1.35±0.14 ab 1.61±0.06 ab 1.83±0.04 a 1.15±0.12 abc 0.88±0.03 abcd 0.96±0.01 abcd 0.99±0.02 abcd < :3n ± ±0.09 a 0.73±0.06 ab 0.62±0.03 ab 0.34±0.29 b 0.50±0.01 ab 0.63±0.07 ab 0.72±0.06 ab 0.64±0.02 ab 0.63±0.02 ab 0.70±0.05 ab 0.86±0.20 a 0.66±0.02 ab 0.55±0.03 ab :5n ± ±0.12 g 6.88±0.61 d 10.9±1.02 ab 12.3±0.14 a 13.2±0.57 a 2.94±0.17 fg 4.35±0.44 ef 3.87±0.01 efg 4.53±0.08 e 4.59±0.43 e 7.14±0.06 d 8.47±0.23 cd 9.74±0.23 bc < :5n ± ±0.02 abcd 1.34±0.29 abc 1.64±0.09 ab 1.72±0.00 ab 1.82±0.05 a 0.55±0.00 cd 0.69±0.16 cd 0.54±0.00 cd 0.44±0.18 d 0.84±0.11 bcd 1.00±0.01 abcd 1.07±0.03 abcd 1.06±0.03 abcd < :6n ± ±0.05 g 9.67±0.56 f 11.1±0.50 ef 13.1±0.36 def 13.1±0.80 def 12.3±0.03 ef 16.7±2.10 bcd 20.1±0.42 ab 24.3±0.48 a 13.0±1.81 def 15.0±0.49 cde 17.6±0.34 bc 18.3±0.53 bc < :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table S3b. Fatty acid (FA) composition (% of total identified) in the phosphatidylethanolamine (E) fraction in skin of fish at 400 g weight (mean±se; n=3 for CC, 0% EA+DHA, and 2% EA and/or DHA, n=2 for other groups 1 ). FA CC 0% EA+DHA 0.5% 1.0% 1.5% 2.0% 0.5% 1.0% 1.5% 2.0% 0.5% 1.0% 1.5% 2.0% 12:0 1.65± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 0.33± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 6.40± ±0.37 c 5.61±0.31 bc 5.73±0.37 abc 6.77±0.18 a 6.43±0.33 abc 5.81±0.09 abc 5.58±0.36 c 5.97±0.16 abc 6.24±0.34 abc 5.61±0.16 bc 5.99±0.07 abc 6.33±0.07 abc 6.73±0.49 ab :0 4.71± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ±0.26 a 2.58±0.05 ab 1.69±0.01 abc 1.17±0.06 bcde 0.99±0.10 cde 2.17±0.04 abc 1.90±0.56 abc 1.07±0.07 cde 0.79±0.06 e 1.01±1.43 cde 1.64±0.02 abcd 1.22±0.02 abcde 0.86±0.02 de < :4n ± ±0.50 a 12.4±0.56 bc 9.19±0.24 def 6.76±0.12 gh 6.28±0.32 h 13.8±0.30 b 10.3±1.32 cde 8.39±1.07 efg 7.63±0.53 fgh 11.4±0.94 cd 8.77±0.08 ef 7.56±0.09 fgh 6.57±0.49 gh < :5n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ±0.04 b 0.45±0.03 ab 0.40±0.03 b 0.35±0.06 b 0.40±0.02 ab 0.37±0.11 b 0.48±0.01 ab 0.47±0.03 ab 0.48±0.05 ab 0.63±0.13 a 0.48±0.07 ab 0.45±0.02 ab 0.45±0.06 ab :5n ± ±0.25 de 5.38±0.67 c 7.81±0.67 ab 8.06±0.38 a 9.33±0.14 a 2.46±0.22 e 3.27±0.60 de 3.01±0.15 de 3.10±0.05 de 3.55±0.14 d 5.23±0.18 c 5.81±0.43 c 6.45±0.16 bc < :5n ± ±0.15 bcd 2.01±0.37 abc 2.18±0.25 ab 2.19±0.09 ab 2.58±0.08 a 0.66±0.06 ef 0.97±0.45 def 0.62±0.05 f 0.55±0.07 f 1.02±0.26 def 1.22±0.04 de 1.21±0.06 de 1.31±0.12 cd < :6n ± ±0.29 f 18.4±0.56 e 20.6±1.22 de 22.1±0.34 bcde 23.6±0.72 bcd 20.4±0.70 de 25.0±2.69 bcd 27.0±0.65 ab 30.5±2.27 a 21.1±3.48 cde 24.3±0.56 bcd 25.9±0.44 abc 27.2±0.52 ab < :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table S3c. Fatty acid (FA) composition (% of total identified) in the phosphatidylserine (S) fraction in skin of fish at 400 g weight (mean±se; n=3 for CC, 0% EA+DHA, and 2% EA and/or DHA, n=2 for other groups 1 ). FA CC 0% EA+DHA 0.5% 1.0% 1.5% 2.0% 0.5% 1.0% 1.5% 2.0% 0.5% 1.0% 1.5% 2.0% 12:0 2.22± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 0.34± ±0.39 ab 0.10±0.06 ab 0.04±0.06 b 0.14±0.01 ab 0.19±0.05 ab 0.55±0.19 ab 0.39±0.40 ab 0.28±0.08 ab 0.22±0.23 ab 1.36±0.61 a 0.38±0.20 ab 0.34±0.26 ab 0.28±0.14 ab :0 10.3± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 22.9± ± ± ± ± ± ± ± ± ± ± ± ± ±

8 16:1n ± ±0.08 ab 0.37±0.15 ab 0.27±0.11 ab 0.21±0.03 ab 0.23±0.07 ab 0.33±0.07 ab 0.42±0.09 ab 0.52±0.04 ab 0.37±0.25 ab 0.73±0.58 a 0.53±0.45 ab 0.21±0.03 ab 0.08±0.14 b :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ±0.20 a 2.56±0.03 ab 1.65±0.05 bcd 1.26±0.03 cd 0.95±0.10 d 2.25±0.08 abc 1.77±0.74 bcd 0.92±0.37 d 0.83±0.13 d 1.70±0.63 bcd 1.54±0.09 bcd 1.34±0.02 cd 1.10±0.27 d < :4n ± ±0.26 a 4.45±0.13 bc 3.26±0.19 bc 2.92±0.15 c 2.84±0.03 c 4.82±0.11 ab 3.66±0.70 bc 4.25±0.67 bc 3.14±0.26 bc 3.72±1.36 bc 3.27±0.13 bc 3.78±0.09 bc 3.19±0.50 bc < :5n ± ±2.03 a 0.66±0.76 b 1.17±0.03 ab 0.93±0.15 ab 0.83±0.06 ab 3.71±0.10 a 2.85±0.23 a 3.12±0.34 a 3.42±0.28 a 1.62±0.57 ab 2.38±0.29 ab 2.49±0.07 a 1.74±0.98 ab :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :5n ± ±0.05 b 1.78±0.29 ab 2.81±0.04 ab 3.05±0.33 ab 3.60±0.32 a 1.44±0.15 ab 1.50±0.96 ab 2.45±1.61 ab 1.94±0.63 ab 1.22±0.30 b 2.12±0.40 ab 2.56±0.77 ab 2.49±0.06 ab :5n ± ±0.35 abcd 3.77±0.14 abcd 4.65±0.11 ab 4.45±0.07 abc 5.31±0.59 a 1.55±0.07 ef 1.45±0.64 ef 1.46±0.71 ef 1.08±0.13 f 1.98±1.24 def 2.48±0.15 bcde 2.44±0.18 cdef 2.30±0.11 def < :6n ± ±1.07 c 23.0±2.23 bc 27.0±0.44 abc 28.4±2.06 ab 30.0±0.82 ab 26.1±0.51 abc 27.1±0.10 abc 30.3±3.12 ab 32.6±4.13 a 23.0±6.17 bc 29.4±2.78 ab 31.3±0.64 ab 29.3±2.66 ab :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table S3d. Fatty acid (FA) composition (% of total identified) in the phosphatidylinositol (I) fraction in skin of fish at 400 g weight (mean±se; n=3 for CC, 0% EA+DHA, and 2% EA and/or DHA, n=2 for other groups 1 ). FA CC 0% EA+DHA 0.5% 1.0% 1.5% 2.0% 0.5% 1.0% 1.5% 2.0% 0.5% 1.0% 1.5% 2.0% 12:0 3.97± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 0.82± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 6.16± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 27.6± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ±0.10 a 2.41±0.07 a 1.91±0.07 abc 1.70±0.08 abcd 1.27±0.26 bcd 2.10±0.40 a 2.14±0.19 a 1.60±0.07 abcd 1.09±0.28 d 2.30±0.45 a 2.05±0.23 ab 1.65±0.00 abcd 1.23±0.09 cd < :4n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :5n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :5n ± ±0.05c 1.51±0.08 abc 2.09±0.33 abc 2.95±0.07 a 3.26±0.58 a 0.75±0.08 c 1.03±0.08 bc 1.07±0.04 bc 1.63±1.13 abc 1.49±0.63 abc 1.55±0.24 abc 2.30±0.55 abc 2.61±0.49 ab :5n ± ±0.08 ab 1.09±0.25 ab 1.16±0.08 ab 1.23±0.02 ab 1.99±0.92 a 0.56±0.35 b 0.56±0.05 b 0.45±0.16 b 0.69±0.40 b 0.63±0.03 b 0.66±0.03 b 0.84±0.23 ab 0.75±0.10 ab :6n ± ±0.21 b 6.41±0.11 ab 6.46±0.14 ab 7.42±0.45 ab 7.44±1.31 ab 7.74±1.26 ab 9.12±1.09 ab 14.3±5.09 a 16.2±9.80 a 7.21±0.44 ab 8.48±0.40 ab 8.75±0.35 ab 9.33±0.68 ab :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± Each statistical replicate originated from a pooled sample of skin from five fish.

9 values calculated by one-way ANOVA test or Mann-Whitney test within the dietary groups except CC. a-h Different superscript letters indicate significant differences between the diets (Tukey s test. <0.05). Table S4a. Fatty acid (FA) composition (% of total identified) in the phosphatidylcholine (C) fraction in skin of fish at 200 g weight (mean±se; n=3 for CC, 0% EA+DHA, and 2% EA and/or DHA, n=2 for other groups 1 ). FA CC 0% EA+DHA 0.5% 1.0% 1.5% 2% 0.5% 1.0% 1.5% 2% 0.5% 1.0% 1.5% 2% 12:0 0.34± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 1.33± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 29.5± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 3.74± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ±0.74 a 22.4±0.52 ab 22.5±0.12 ab 22.9±1.18 ab 20.8±0.48 b 22.6±1.13 ab 21.6±0.71 ab 23.1±0.26 ab 20.8±0.58 b 22.3±0.29 ab 20.6±0.56 b 23.1±2.01 ab 21.7±0.34 ab :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ±0.29 a 6.84±0.17 abc 6.22±0.03 bcd 4.86±0.32 ef 4.49±0.29 f 7.87±0.32 a 5.99±0.12 cde 5.42±0.26 def 5.33±0.03 def 7.53±0.21 ab 6.34±0.04 bcd 4.92±0.08 ef 4.60±0.19 f < :3n ± ±0.05 a 0.49±0.00 b 0.32±0.01 c 0.21±0.00 de 0.15±0.01 e 0.53±0.03 b 0.30±0.01 cd 0.27±0.02 cd 0.16±0.00 e 0.51±0.03 b 0.31±0.02 c 0.26±0.01 cd 0.17±0.01 e < :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ±0.12 a 2.51±0.16 ab 1.63±0.14 cd 0.81±0.28 e 0.77±0.04 e 2.34±0.33 abc 1.65±0.03 bcd 1.10±0.13 de 0.83±0.02 e 2.45±0.10 abc 1.89±0.16 abc 1.06±0.12 de 0.80±0.04 e < :4n ± ±0.31 a 5.92±0.35 b 3.97±0.30 cdef 2.89±0.10 f 2.84±0.07 f 5.65±0.91 bc 5.06±0.08 bcd 3.62±0.31 def 3.42±0.19 def 5.60±0.45 bc 4.57±0.29 bcde 3.23±0.45 ef 2.99±0.15 ef < :5n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :5n ± ±0.18 g 5.63±0.03 cdef 9.95±0.86 ab 9.04±1.44 abc 11.0±0.95 a 2.81±0.34 g 3.34±0.27 fg 3.22±0.34 fg 3.55±0.26 efg 4.18±0.30 defg 6.66±0.13 bcd 6.22±1.05 cde 7.80±0.57 abc < :5n ± ±0.05 defg 1.14±0.04 abc 1.60±0.07 a 1.32±0.16 ab 1.56±0.07 a 0.50±0.08 efg 0.43±0.05 fg 0.43±0.06 g 0.42±0.02 g 0.80±0.01 cde 0.88±0.07 bcd 0.76±0.12 cdef 0.84±0.07 cde < :6n ± ±0.62c 8.57±0.76 bc 10.8±0.52 abc 8.66±0.91 bc 11.7±0.55 ab 10.3±1.67 bc 14.9±1.77 ab 15.7±2.44 ab 18.5±1.77 a 9.26±1.19 bc 13.6±0.63 ab 12.9±2.27 ab 14.8±1.25 ab < :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table S4b. Fatty acid (FA) composition (% of total identified) in the phosphatidylethanolamine (E) fraction in skin of fish at 200 g weight (mean±se; n=3 for CC, 0% EA+DHA, and 2% EA and/or DHA, n=2 for other groups 1 ). FA CC 0% EA+DHA 0.5% 1.0% 1.5% 2% 0.5% 1.0% 1.5% 2% 0.5% 1.0% 1.5% 2% 12:0 0.84± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 0.39± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 7.49± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 6.03± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 18:1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ±0.03 b 1.01±0.01 ab 0.95±0.01 ab 1.13±0.01 ab 1.04±0.10 ab 1.06±0.08 ab 1.06±0.07 ab 1.19±0.06 a 1.25±0.08 a 1.10±0.07 ab 1.11±0.02 ab 1.19±0.10 a 1.11±0.04 ab :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ±0.02 a 0.38±0.02 b 0.29±0.03 bcd 0.25±0.00 de 0.23±0.02 de 0.38±0.01 b 0.26±0.02 cde 0.25±0.02 de 0.22±0.01 de 0.35±0.02 bc 0.30±0.02 bcd 0.26±0.01 cde 0.20±0.01 e < :2n ± ±0.17 b 0.96±0.09 ab 0.85±0.27 ab 1.12±0.01 ab 1.06±0.09 ab 0.97±0.09 ab 1.16±0.02 a 1.23±0.06 a 1.33±0.04 a 1.14±0.02 ab 1.20±0.01 a 1.15±0.03 ab 1.15± :3n ± ±0.13 a 2.04±0.04 a 1.45±0.03 bc 1.01±0.00 de 0.86±0.04 e 1.78±0.09 ab 1.32±0.03 cd 0.88±0.07 e 0.70±0.04 e 1.89±0.09 ab 1.50±0.12 bc 0.93±0.11 e 0.72±0.03 e < :4n ± ±0.79 a 11.2±0.64 b 7.80±0.27 def 5.95±0.24 fg 5.52±0.20 g 11.1±1.20 bc 9.21±0.26 bcd 6.89±0.43 defg 6.37±0.20 efg 10.8±0.63 bc 8.49±0.34 cde 6.25±0.37 efg 5.53±0.26 g < :5n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :5n ± ±0.25 f 5.45±0.10 de 7.82±0.80 abc 8.09±0.09 ab 9.03±0.47 a 2.92±0.18 f 3.07±0.38 f 2.98±0.09 f 3.15±0.21 f 4.12±0.30 ef 5.61±0.12 cde 5.48±0.54 de 6.24±0.37 bcd < :5n ± ±0.14 bc 1.69±0.00 ab 2.38±0.02 a 2.10±0.08 a 2.26±0.11 a 0.65±0.03 de 0.53±0.11 e 0.52±0.04 e 0.49±0.04 e 1.17±0.02 bc 1.13±0.10 bcd 1.07±0.13 cd 1.08±0.14 bcd < :6n ± ±1.82 b 17.1±1.39 ab 20.7±0.73 ab 17.8±0.58 ab 20.2±1.78 ab 18.3±1.83 ab 22.7±1.99 a 23.8±3.79 a 24.9±2.04 a 17.2±2.84 ab 21.6±0.89 ab 22.2±3.65 ab 22.2±2.98 ab :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table S4c. Fatty acid (FA) composition (% of total identified) in the phosphatidylserine (S) fraction in skin of fish at 200 g weight (mean±se; n=3 for CC, 0% EA+DHA, and 2% EA and/or DHA, n=2 for other groups 1 ). FA CC 0% EA+DHA 0.5% 1.0% 1.5% 2% 0.5% 1.0% 1.5% 2% 0.5% 1.0% 1.5% 2% 12:0 0.74± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 0.73± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 17.4± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 17.6± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ±0.13 a 2.49±0.11 a 1.47±0.06 bc 1.01±0.02 bcde 0.77±0.06 de 1.68±0.20 ab 1.23±0.21 bcde 0.91±0.05 cde 0.83±0.11 cde 1.42±0.36 bcd 1.45±0.17 bc 1.03±0.05 bcde 0.73±0.02 e < :4n ± ±0.19 a 5.52±1.1 0ab 3.09±0.09 bc 2.83±0.70 c 2.80±0.56 c 4.29±0.48 abc 3.59±0.60 bc 3.18±0.18 bc 2.73±0.19 c 3.72±0.06 bc 3.15±0.25 bc 2.86±0.24 c 2.27±0.15 c < :5n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :5n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :5n ± ±0.65 ab 3.23±0.78 ab 4.87±0.39 a 2.47±0.77 ab 3.93±0.93 ab 1.38±0.22 b 1.13±0.13 b 1.02±0.21 b 1.03±0.14 b 1.99±0.43 ab 2.44±0.14 ab 1.94±0.64 ab 1.99±0.29 ab :6n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ±

11 Table S4d. Fatty acid (FA) composition (% of total identified) in the phosphatidylinositol (I) fraction in skin of fish at 200 g weight (mean±se; n=3 for CC, 0% EA+DHA, and 2% EA and/or DHA, n=2 for other groups 1 ). FA CC 0% EA+DHA 0.5% 1.0% 1.5% 2% 0.5% 1.0% 1.5% 2% 0.5% 1.0% 1.5% 2% 12:0 1.85± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 0.32± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 9.89± ± ± ± ± ± ± ± ± ± ± ± ± ± :0 24.6± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :2n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ±0.07 a 1.87±0.19 abc 1.66±0.22 abcd 1.17±0.09 bcd 0.97±0.09 d 1.80±0.10 abcd 1.43±0.10 abcd 0.98±0.23 d 0.94±0.13 d 1.99±0.07 ab 1.76±0.08 abcd 1.14±0.26 bcd 1.08±0.17 cd < :4n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :5n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :5n ± ±0.14 c 2.42±0.17 abc 3.49±0.01 ab 3.18±0.53 ab 4.99±1.02 a 0.99±0.04 c 1.94±0.42 bc 1.04±0.11 c 1.14±0.29 c 1.52±0.30 bc 1.83±0.20 bc 2.33±0.54 abc 2.66±0.24 abc < :5n ± ±0.63 ab 1.53±0.53 ab 1.06±0.11 ab 2.56±1.51 a 1.73±0.27 ab 0.16±0.16 b 0.75±0.35 ab 0.45±0.09 ab 0.58±0.04 ab 1.29±0.59 ab 0.59±0.02 ab 1.23±0.53 ab 1.18±0.25 ab :6n ± ± ± ± ± ± ± ± ± ± ± ± ± ± :3n ± ± ± ± ± ± ± ± ± ± ± ± ± ± Each statistical replicate originated from a pooled sample of skin from five fish. values calculated by one-way ANOVA or Mann-Whitney test within the dietary groups except CC. a-g Different superscript letters indicate significant differences between diets (Tukey s test. <0.05).