Zebrafish feed composition.

1<p>MP Biomedical, Solon, OH, USA, cat. no. 02901293;</p>2<p>Idun Industri AS, Skjetten, Norway;</p>3<p>Fluka Chemie, Buchs, Switzerland cat. no. 31400;</p>4<p>Sigma-Aldrich, St Louis, MO, USA;</p>5<p>Møllers tran, Oslo, Norway;</p>6<p>DSM nutritional products, Oslo, Norway;</p>7<p>Eldorado, Oslo, Norway;</p>8<p>Carophyll Pink, Hoffman-La Roche, Basle, Switzerland;</p><p>*Details on mineral mix, vitamin mix and amino acid mix composition can be found in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089347#pone-0089347-t001" target="_blank">tables 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089347#pone-0089347-t002" target="_blank">2</a> and 3.</p

Gene expression profile and Mmp activity of ontogenetic scales after feeding the different diets.

<p><b><i>A</i></b><i>:</i> Gene expression analysis of ontogenetic scales from fish fed the low ARA diet (black bars) or the high ARA diet (white bars). No statistically different effects of the different diets were observed, not even in <i>osteocalcin</i> expression (P = 0.900; non-parametric test). Transcript abundance is expressed relative to an index of the reference genes <i>rpl13a</i> and <i>tuba1</i>. Results are displayed as means ± SEM of 16 scales randomly obtained from all of the 4 replicate tanks. <b><i>B</i></b>: Example of a Coomassie-stained gelatin gel showing increased MMP activity in ontogenetic scales from the high ARA group (two lanes to the right) compared to the low ARA group (left lanes). Protein mass ladder is given in kiloDalton (kDa). <b><i>C</i></b>: Semiquantitative analysis of gelatinolytic activity demonstrates that total of Mmp2 and Mmp9 activity, secreted by cultured ontogenetic scales, is higher in scales from fish fed the high ARA diet compared to the low ARA diet. The total amount of secreted gelatinolytic activity is significantly increased in the high ARA group (P<0.05; One sample T test). Results of high ARA samples are expressed relative to low ARA samples analyzed on the same gel. Bars represent the mean ± SEM of 8 samples.</p

In-vitro exposure of scales to arachidonic acid.

<p>Semiquantitative analysis of gelatinolytic matrix metalloproteinases of culture medium after <i>in vitro</i> exposure of ARA for 24 hours (<b><i>A</i></b> and <b><i>B</i></b>) and 48 hours (<b><i>C</i></b> and <b><i>D</i></b>). <b><i>A</i></b>: ARA has no effect on Mmp2 activity after 24 hours of culture. <b><i>B</i></b>: A transient, non-significant increasing trend is measured in Mmp9 activity with increasing ARA concentration. <b><i>C</i></b>: Stimulatory effects of ARA on Mmp2 are not significant. <b><i>D</i></b>: ARA significantly increases MMP9 after 48 hours of culture, with a concentration-dependent trend. Activities of Mmp2 (<b><i>A</i></b>, <b><i>C</i></b>) and Mmp9 (<b><i>B</i></b>, <b><i>D</i></b>) are presented relative to the Mmp activity of samples taken from control cultures. Samples are expressed as mean ± SEM of 2 (24 h) or 6 replicates (48 h). Statistical analysis was done with the Student's <i>t</i>-test or Mann-Whitney U-test when results were not normally distributed (* p<0,05, ** p<0,01).</p

Effects of the different diets on scale mineralization.

<p>Representative examples of scales on which the quantifications from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089347#pone-0089347-g001" target="_blank">Figure 1</a> were based. <b><i>A</i></b>: Example of a scale from the low ARA group stained for mineralization (Von Kossa staining, calcium phosphates stained brown). The scale is almost completely mineralized and the brown staining is only absent in the focus (lower central region) of the scale. Bar = 250 µm. <b><i>B</i></b>: Detailed image of the focus of the scale shown in (<b><i>A</i></b>) with several unmineralized small resorption pits where staining is absent (arrows). Bar = 100 µm. <b><i>C</i></b>: TRAcP activity-stained ontogenetic scale from the low ARA group with few red-stained areas positive for TRAcP activity, indicated by arrows. Bar = 250 µm. <b><i>D</i></b>: Von Kossa stained ontogenetic scale from the high ARA group. Arrows indicate resorption pits outside the focus, seen more often in scales from high than from low ARA fed fish. Bar = 250 µm. <b><i>E</i></b>: Detailed image of the scale shown in (<b><i>D</i></b>) with more resorption pits with typical round edges as a result of osteoclastic matrix degradation. Bar = 100 µm. <b><i>F</i></b>: TRAcP activity-stained ontogenetic scale from the high ARA group with more spots stained red for TRAcP activity than in the low ARA group, indicating the presence of more osteoclasts in this group. Bar = 250 µm.</p

Effects of the different diets on scale mineralization.

<p>Analyses of the mineralized layer of ontogenetic scales were done after feeding the low ARA diet (solid dots) or the high ARA diet (open dots) for 4 weeks. <b><i>A</i></b>: Calcium:phosphorus molar ratios were significantly lower in high ARA fed fish. <b><i>B</i></b>: Surface area of the scales was unaffected. <b><i>C</i></b>: The number of resorption pits per individual scale was higher in the high ARA fed fish. <b><i>D</i></b>: Indeed, total perimeter of resorption pits also increased. <b><i>E</i></b>: Percentage of each scale that is demineralized (as a result of osteoclast activity) was also increased in high ARA fed fish. <b><i>F</i></b>: Number of TRAcP-stained regions per scale. Each replicate is shown; the horizontal lines represent means of 36 replicates. Statistical analysis was done with the Students T-test (*** p<0.001) except for (<b><i>E</i></b>) which required non-parametric testing (Mann Witney-U test, ** p<0.01).</p

Analysis of scale mineralization during regeneration.

<p><b><i>A</i></b>: Absolute calcium contents in regenerating scales sampled at different time points after scale removal. Calcium content increases similarly in both groups as the scale grows during regeneration. <b><i>B</i></b>: Calcium:phosphorus molar ratio of regenerating scales of different ages. The ratio increases in both groups from day 4 to 7, indicative of a changed mineral composition on the scale matrix. Effect of the diet is only observed on day 7; the Ca:P ratio in the high ARA groups is significantly increased compared to the low ARA group. Samples are expressed as mean ± SEM (N = 9); black bars represent samples obtained from fish fed the low ARA diet, and white bars represent samples from fish fed the high ARA diet. Statistical testing was conducted with the Students T-test (* p<0.05).</p

Gene expression profile and Mmp activity of regenerating scales of fish fed the different diets.

<p><b><i>A</i></b>: Gene expression of <i>sp7</i> and <i>rankl</i> (osteoblast), as well as <i>mmp9</i> (osteoclast) is higher in the high ARA group compared to the low ARA group in 4-day-old regenerating scales. <b><i>B</i></b>: After 7 days of scale regeneration, these differences mostly disappeared. Gene expression of the low ARA group has increased compared to expression levels at day 4 and expression of <i>rankl</i> is also significantly higher compared to the high ARA group. Gene expression is shown as mean normalized expression (MNE). MNE for scales obtained from fish fed the ARA-restricted diet (low ARA) are shown with black bars and for scales from fish fed the ARA-enriched diet (high ARA) with white bars. Bars represent the mean ± SEM of 9 replicates. After assessment of normal distribution (D'Agostino & Pearson normality test), statistical analysis was done with either Student's <i>t</i>-test, or Mann-Whitney U-test (* p<0.05, ** p<0.01). <b><i>C</i></b>: Enzymatic Mmp9 activity of 7-day-old regenerating scales is higher in the high ARA group, as seen on gelatin zymography. Protein mass ladder is depicted in kiloDalton (kDa). <b><i>D</i></b>: Semiquantitative analysis of enzymatic Mmp2 and Mmp9 activity and total Mmp activity in 4-day-old and 7-day-old regenerating scales shows the increased Mmp9 activity observed in (<b><i>C</i></b>). Mmp activity of scales from high ARA fish is expressed relative to those of low ARA fish analyzed on the same gel. Bars represent the mean of 4 samples; horizontal line represents the low ARA samples. One sample <i>t</i>-test was used to compare the mean Mmp activity to 1. P-values for 4 days regeneration: P = 0.07 for Mmp2, P = 0.22 for Mmp9 and P = 0.06 for total. P-values for 7 days regeneration: P = 0.72 for Mmp2, P = 0.05 for Mmp9 and P = 0.29 for total.</p

Growth parameters analysed: D21 length (n = 48, F_(3,40) = 1.09, p = 0.3641), D21 weight (n = 48, F_(3,40) = 0.71, p = 0.5516), D21 condition factor (n = 48, F_(3,40) = 5.29, p = 0.0036), D42 length (n = 47, F_(3,39) = 1.30, p = 0.2884), D42 weight (n = 47, F_(3,39) = 0.90, p = 0.4500), and D42 condition factor (n = 47, F_(3,39) = 7.37, p = 0.0005).

<p>* = p<0.05,</p><p>** = p<0.01,</p><p>*** = p<0.001</p><p>Growth parameters analysed: D21 length (n = 48, F_(3,40) = 1.09, p = 0.3641), D21 weight (n = 48, F_(3,40) = 0.71, p = 0.5516), D21 condition factor (n = 48, F_(3,40) = 5.29, p = 0.0036), D42 length (n = 47, F_(3,39) = 1.30, p = 0.2884), D42 weight (n = 47, F_(3,39) = 0.90, p = 0.4500), and D42 condition factor (n = 47, F_(3,39) = 7.37, p = 0.0005).</p

Optimized UPLC-MS/MS conditions per compound.

<p>* = quantifier ion</p><p>Optimized UPLC-MS/MS conditions per compound.</p

Cortisol (pmol per scale) in ontogenetic scales after 21 and 42 days of treatment (CCα = decision limit and CCβ = detection capability were calculated using an average weight of 0.026 g per scale).

<p>One value (2.8 pmol per scale) of STRESS day 42 was omitted in the scatter/boxplot for reasons of presentation. Error bars in scatter/boxplots are defined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123411#pone.0123411.g001" target="_blank">Fig 1</a>. For statistics, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123411#pone.0123411.s003" target="_blank">S2 Table</a>.</p