Detection of malformations in sea urchin plutei exposed to mercuric chloride using different fluorescent techniques

Isabella Buttino, Jiang-Shiou Hwang, Giovanna Romano, Chi-Kuang Sun, Tzu-Ming Liu, David Pellegrini, Andrea Gaion, Davide Sartori

Istituto Superiore per la Protezione e la Ricerca Ambientale, ISPRA_ STS-Livorno, Piazzale dei marmi 12, 57123, Italy.

Abstract

Embryos of Mediterranean sea urchin Paracentrotus lividus and subtropical Echinometra mathaei were exposed to 5,10, 15 and 20 µg L^-1, and to 1, 2, 3 and 4 µg L^-1 mercuric chloride (HgCl₂), respectively. The effective concentration (EC50) inducing malformation in 50% of 4-arm pluteus stage (P4) was 16.14 µg L^-1 for P. lividus and 2.41 µg L^-1 for E. mathaei. Two-photon (TP), second (SHG) and third harmonic generation (THG) microscopy techniques, TUNEL staining, propidium iodide (PI) and Hoechst 33342 probes were used to detect light signals or to stain apoptotic and necrotic cells in fixed and alive plutei. Signals were detected differently in the two species: TP fluorescence, commonly associated with apoptotic cells, did not increase with increasing HgCl₂ concentrations in P. lividus and in fact, the TUNEL did not reveal induction of apoptosis. PI fluorescence increased in P. lividus in a dose-dependent manner, suggesting a loss of cell permeability. In E. mathaei plutei TP fluorescence increased at increasing HgCl₂ concentrations. THG microscopy revealed skeletal rods in both species. Different fluorescent techniques, used in this study, are proposed as early-warning systems to visualize malformations and physiological responses in sea urchin plutei.

Keywords: Harmonic generation microscopy; Two-photon microscopy; Confocal microscopy; Skeletal rod; Apoptosis.