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Int J Mol Sci
2020 Jan 22;213:. doi: 10.3390/ijms21030719.
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Combined Effects of Diatom-Derived Oxylipins on the Sea Urchin Paracentrotus lividus.
Esposito R
,
Ruocco N
,
Albarano L
,
Ianora A
,
Manfra L
,
Libralato G
,
Costantini M
.
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Oxylipins are diatom-derived secondary metabolites, deriving from the oxidation of polyunsatured fatty acids that are released from cell membranes after cell damage or senescence of these single-celled algae. Previous results revealed harmful toxic effects of polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) on sea urchin Paracentrotus lividus embryonic development by testing individual compounds and mixtures of the same chemical group. Here, we investigated the combined effects of these compounds on sea urchin development at the morphological and molecular level for the first time. Our results demonstrated that oxylipin mixtures had stronger effects on sea urchin embryos compared with individual compounds, confirming that PUAs induce malformations and HEPEs cause developmental delay. This harmful effect was also confirmed by molecular analysis. Twelve new genes, involved in stress response and embryonic developmental processes, were isolated from the sea urchin P. lividus; these genes were found to be functionally interconnected with 11 genes already identified as a stress response of P. lividus embryos to single oxylipins. The expression levels of most of the analyzed genes targeted by oxylipin mixtures were involved in stress, skeletogenesis, development/differentiation, and detoxification processes. This work has important ecological implications, considering that PUAs and HEPEs represent the most abundant oxylipins in bloom-forming diatoms, opening new perspectives in understanding the molecular pathways activated by sea urchins exposed to diatom oxylipins.
Figure 1. Sea urchin Paracentrotus lividus exposed before fertilization to different concentrations of mixtures of polyunsaturated aldehydes (PUA) + hydroxyacids (HEPE). Percentage of normal, malformed, early, delayed, and shortened plutei and gastrulae in controls (embryos grown in the absence of PUA/HEPE mixtures) and treated samples are reported. Five experiments are numbered on the top of the histogram. For the numerical data of the five experiments see also Supplementary Table S1. The statistical significance between different groups is reported as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 2. (A) Interactomic analysis by STRING (https://string-db.org/). The network graphically displays the relationship between genes. The biological relationships between genes are indicated by different colors. Known interactions: reported by database = light blue and determined experimentally = pink. Expected interactions: gene proximity = green; gene fusion = red; genes with similar pattern = light blue. (B) Homo sapiens gene names and the corresponding P. lividus orthologous genes. The new genes isolated in the present work are underlined.
Figure 3. Real-time qPCR at the blastula (5 hpf), gastrula (21 hpf), and pluteus (48 hpf) stages of the sea urchin P. lividus. Histograms show the fold-changes of 23 genes involved in different functional processes: stress (A), skeletogenesis (B), development/differentiation (C), and detoxification (D). Fold differences greater than ± 1.5 (see red dotted horizontal guidelines at values of +1.5 and −1.5) were considered significant.
Figure 4. Heatmaps (Heatmapper available at www.heatmapper.ca) showing the expression profiles and hierarchical clustering of 23 genes analyzed by real-time qPCR in P. lividus embryos at the three different developmental stages (blastula, gastrula, and pluteus) treated with mixtures decadienal 0.3 μM, heptadienal 0.7 μM, and octadienal 1.0 μM, and HEPEs 1.6 μM. Colour code: red, up-regulated genes respect to the control (embryos grown without PUAs + HEPEs mixtures); blue, down-regulated genes.
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