PAHs and PCBs Affect Functionally Intercorrelated Genes in the Sea Urchin Paracentrotus lividus Embryos

Abstract

Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) represent the most common pollutants in the marine sediments. Previous investigations demonstrated short-term sublethal effects of sediments polluted with both contaminants on the sea urchin Paracentrotus lividus after 2 months of exposure in mesocosms. In particular, morphological malformations observed in P. lividus embryos deriving from adults exposed to PAHs and PCBs were explained at molecular levels by de novo transcriptome assembly and real-time qPCR, leading to the identification of several differentially expressed genes involved in key physiological processes. Here, we extensively explored the genes involved in the response of the sea urchin P. lividus to PAHs and PCBs. Firstly, 25 new genes were identified and interactomic analysis revealed that they were functionally connected among them and to several genes previously defined as molecular targets of response to the two pollutants under analysis. The expression levels of these 25 genes were followed by Real Time qPCR, showing that almost all genes analyzed were affected by PAHs and PCBs. These findings represent an important further step in defining the impacts of slight concentrations of such contaminants on sea urchins and, more in general, on marine biota, increasing our knowledge of molecular targets involved in responses to environmental stressors.

Keywords: aromatic hydrocarbons; polychlorinated biphenyls; sea urchin.

Figure 1

Real-time qPCR at the pluteus (48 hpf) stages of the sea urchin P. lividus embryos, deriving from adults exposed for 2 months to sediments contaminated with PAHs and PCBs. Data are reported as a fold difference compared with control embryos, deriving from adults reared in tanks with non-contaminated sediments (mean ± SD). Histograms show the fold-changes of 25 genes involved in three functional processes: stress response, development/differentiation and detoxification. Fold differences greater than ±1.5 (see red dotted horizontal guidelines at values of +1.5 and −1.5) were considered significant. Real-time qPCR reactions were carried out in triplicate. Statistical differences were evaluated by nonparametric Mann–Whitney test. p-values < 0.05 were considered significant.

Figure 2

Gene networks obtained by STRING interactome analysis of ten genes involved in stress response and detoxification. Correlation confidence score cut-off of 400 was reported. Among functionally correlated genes, those with up (red) and down (green) expression affected by PAHs (a) and PCBs (b) were reported. Color shading depends on fold-change values. Gray spheres represent additional connections. The list of human orthologous genes used for Network analysis is reported in Supplementary Table S2.

Figure 3

Gene network obtained by STRING interactome analysis of 15 genes involved in developmental processes. Correlation confidence score cut-off of 400 was reported. Among functionally correlated genes, those with up (red), down (green) and unchanged (blue) expression affected by PAHs (a) and PCBs (b) were reported. Color shading depends on fold-change values. Gray spheres represent additional connections. The whole list of human orthologous genes used for Network analysis is reported in Supplementary Table S2.

Figure 4

Heatmap showing the expression profiles and hierarchical clustering of 25 genes analyzed by Real Time qPCR in embryos deriving from adult P. lividus sea urchins exposed for 2 months to sediment contaminated with PAHs and PCBs. Color code: green, up-regulated genes with respect to the control; red, down-regulated genes with respect to the control; black, genes for which there is no variation in gene expression with respect to the control.