Aquatic Toxicology 158 (2015) 181–191 Contents lists available at ScienceDirect Aquatic Toxicology j ourna l ho me pa ge: www.elsevier.com/locate/aquatox Exposure of Paracentrotus lividus male gametes to engineered nanoparticles affects skeletal bio-mineralization processes and larval plasticity Chiara Gambardella a,∗ , Sara Ferrando b , Silvia Morgana a , Lorenzo Gallus b , Paola Ramoino b , Silvia Ravera c , Mattia Bramini d , Alberto Diaspro e , Marco Faimali a , Carla Falugi f a Institute of Marine Science (ISMAR), National Council of Researches (CNR), Via De Marini 6, 16149 Genova, Italy b Department of Earth, Environment and Life Sciences (DISTAV), University of Genova, Viale Benedetto XV 5, 16136 Genova, Italy c Department of Pharmacy (DIFAR), Biochemistry Lab., University of Genova, Viale Benedetto XV 5, 16136 Genova, Italy d Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia (IIT), Via Morego 30, 16163 Genova, Italy e Department of Nanophysics, Istituto Italiano di Tecnologia (IIT), Via Morego 30, 16163 Genova, Italy f Department of Earth, Environment and Life Sciences (DISVA), Polytechnic University of Marche, Ancona, Italy a r t i c l e i n f o Article history: Received 19 August 2014 Received in revised form 14 November 2014 Accepted 18 November 2014 Available online 25 November 2014 Keywords: Biomineralization Cell migration Nanoparticles Paracentrotus lividus Skeletogenesis Sperm exposure a b s t r a c t The aim of this study is to contribute to the understanding of the mechanisms underlying nanoparticle (NP)-induced embryotoxicity in aquatic organisms. We previously demonstrated that exposure of male gametes to NPs causes non-dose-dependent skeletal damage in sea urchin (Paracentrotus lividus) larvae. In the present study, the molecular mechanisms responsible for these anomalies in sea urchin devel- opment from male gametes exposed to cobalt (Co), titanium dioxide (TiO 2 ) and silver (Ag) NPs were investigated by histochemical, immunohistochemical and Western blot analyses. P. lividus sperm were exposed to different NP concentrations (from 0.0001 to 1 mg/L). The distribution of molecules related to skeletogenic cell identification, including ID5 immunoreactivity (IR), wheat germ agglutinin (WGA) affinity and fibronectin (FN) IR, were investigated by confocal laser scanning microscopy at the gastrula (24 h) and pluteus (72 h) stages. Our results identified a spatial correspondence among PMCs, ID5 IR and WGA affinity sites. The altered FN pattern suggests that it is responsible for the altered skeletogenic cell migration, while the Golgi apparatus of the skeletogenic cells, denoted by their WGA affinity, shows different aspects according to the degree of anomalies caused by NP concentrations. The ID5 IR, a specific marker of skeletogenic cells in sea urchin embryos (in particular of the msp130 protein responsible for Ca 2+ and Mg 2+ mineralization), localized in the cellular strands prefiguring the skeletal rods in the gastrula stage and, in the pluteus stage, was visible according to the degree of mineralization of the skeleton. In conclusion, the present study suggests that the investigated NPs suspended in seawater interfere with the bio-mineralization processes in marine organisms, and the results of this study offer a new series of specific endpoints for the mechanistic understanding of NP toxicity. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Sea urchins are model organisms for the assessment of seawater quality, developmental biology, embryology and bio- mineralization processes (Kobayashi, 1991; Wilt, 2005; Jasny and ∗ Corresponding author. Tel.: +39 0106475429. E-mail addresses: chiagamba@gmail.com, chiara.gambardella@ge.ismar.cnr.it (C. Gambardella). Prunell, 2006; Siller et al., 2013; Gambardella et al., 2014). Cur- rently, they are successfully used by international agencies for the bio-monitoring and health status assessment of sea coastal environments (USEPA, 2002; ASTM, 2004; Pinsino et al., 2008). Paracentrotus lividus is emerging as a valid tool for studies in marine ecotoxicology (Matranga and Corsi, 2012). Because both the gametes and embryos of P. lividus are affected by chemi- cals and xenobiotics dispersed in seawater, several laboratories are currently proposing this alternative and ethical model for the standardization of ecotoxicity tests to identify the noxious http://dx.doi.org/10.1016/j.aquatox.2014.11.014 0166-445X/© 2014 Elsevier B.V. All rights reserved.