Developmental defects induced by gadolinium ions in sea urchin embryos of phylogenetically distant species

Gadolinium (Gd) is nowadays an emergent environmental pollutant: it is a metal of the lanthanide series of the elements whose chelates are commonly employed as contrast agents for magnetic resonance imaging and subsequently released into the aquatic environment. Sea urchin embryos are highly sensitive to several kinds of stressors and able to activate different defense strategies. The aim of this study was to analyze the consequences of embryo exposure to sublethal Gd concentrations. We compared the effects of Gd on the development of four phylogenetically distant sea urchin species: two Mediterranean species, Paracentrotus lividus and Arbacia lixula, and two species living in the East coast of Australia, Heliocidaris tuberculata and Centrostephanus rodgersii. In all these species, we observed a general delay of embryo development at 24 hours post-fertilization (gastrula), and a strong inhibition of skeleton growth at 48 hours (pluteus), frequently displayed by an asymmetrical pattern. Since it was demonstrated that the autophagic and apoptotic processes are important defense mechanisms against stress in P. lividus embryos (1, 2, 3), we further investigated the induction of these two processes. Autophagy in P. lividus embryos was inspected by acridine orange (AO) vital staining, Western Blot analysis of total lysates and LC3 (an autophagic marker) immunofluorescence on whole-mount embryos. Overall, the results showed an increase of the LC3 protein at 24 and 48h and confocal microscopy confirmed the increased number of autophagosomes and autophagolysosomes. In contrast, the study of apoptosis performed by immunofluorescence (IF) staining using an anti-cleaved-caspase-3 antibody on whole-mount embryos after 24h and 48h exposure showed no apoptotic induction. In conclusion, the results showed that Gd highly perturbs skeletogenesis in the four species analyzed, even if with species-specific threshold levels of sensitivity, and that autophagy is a molecular process activated in P. lividus embryos, probably acting as a cell survival strategy to defend the developmental program. Taken together, the results pose serious questions on the hazard of Gd in the marine environment.