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Mar Drugs
2019 Mar 01;173:. doi: 10.3390/md17030144.
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Molecular and Morphological Toxicity of Diatom-Derived Hydroxyacid Mixtures to Sea Urchin Paracentrotus lividus Embryos.
Albarano L
,
Ruocco N
,
Ianora A
,
Libralato G
,
Manfra L
,
Costantini M
.
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Oxylipins such as polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) are signaling molecules derived from the oxidation of polyunsaturated fatty acids. They are common in diatoms that constitute a major group of microalgae in freshwater and oceanic ecosystems. Although HEPEs represent the most common oxylipins produced by diatoms, little information is available on their effects on marine invertebrates, and most of the information has been obtained by testing individual HEPEs. Our previous studies reported that four hydroxyacids, i.e., 5-, 9-, 11-, and 15-HEPE, were able to induce malformations and a marked developmental delay in sea urchin Paracentrotus lividus embryos, which had not been reported for other oxylipins. Here, we tested a mixture of 5-, 9-, 11-, and 15-HEPE at different concentrations for the first time. The results showed that mixtures of HEPEs have synergistic effects that are much more severe compared to those of individual HEPEs: The HEPE mixtures induced malformations in sea urchin embryos at lower concentrations. Increasing HEPE mixture concentrations induced a marked increase in the number of delayed embryos, until all embryos were delayed at the highest concentration tested. At the molecular level, the HEPE mixtures induced variations in the expression of 50 genes involved in different functional processes, mainly down-regulating these genes at the earliest stages of embryonic development. These findings are ecologically significant, considering that during diatom blooms, sea urchins could accumulate HEPEs in concentrations comparable to those tested in the present study.
Figure 2. Photos (taken with Zeiss Axiovert 135TV microscope, 10× magnification, 0.30 numerical aperture) of (A) controls at the pluteus stage (at 48 h post fertilization (hpf); embryos in sea water without HEPE mixtures), (B) malformed gastrula, (C) early plutei, (D–G) malformed plutei after incubation with 5-, 9-, 11-, and 15-HEPE mixtures. Scale bar: 50 µm.
Figure 3. Photos (taken with Zeiss Axiovert 135TV microscope, 10× magnification, 0.30 numerical aperture) of (A) control (embryos in sea water without HEPE mixtures) at one week after fertilization, (B–H) abnormal embryos after incubation with 5-, 9-, 11-, and 15-HEPE mixtures at the concentration of 6.0 and 7.0 μM. Scale bar: 50 µm.
Figure 4. Real-time qPCR at blastula (5 hpf), gastrula (21 hpf), and pluteus (48 hpf) stages. Histograms show the differences in the expression levels of 50 genes involved in different functional processes: stress, skeletogenesis, development/differentiation, and detoxification. Paracentrotus lividus embryos were grown in the presence of 5-, 9-, 11-, and 15-HEPE mixtures at the concentration of 2.8 μM. Fold differences greater than ±2 (see red dotted horizontal guidelines at values of +2 and −2) were considered significant (see Supplementary Table S1 for the values).
Figure 5. Heatmap (Heatmapper, available at the site www.heatmappear.ca) of differentially expressed genes in the three developmental stages (blastula, gastrula, and pluteus) after treatment with a 5-, 9-, 11-, and 15-HEPE mixture at the concentration of 2.8 μM. Color codes: red, negative values of gene expression (down-regulated genes with respect to the control, embryos developed in sea water without HEPE mixture); green, positive values of gene expression (up-regulated genes with respect to the control); black, genes for which there was no variation of expression with respect to the control.
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