Modulation of stress factors for cryopreservation of Paracentrotus lividus (Lamarck 1816) larvae

In the present work we modulated two stress factors salinity and temperature, whose ranges have been previously determined by bioassays using six pre-treatments (18 °C-29.5‰; 18 °C-35‰; 18 °C-39‰ and 20 °C-29.5‰; 20 °C-35‰; 20 °C-39‰), in order to obtain a successful cryopreservation protocol for pluteus larvae of the sea urchin P. lividus (Lamarck 1816). Toxicity tests were performed with different cryoprotectants in a range of 0.5-3 M. Best results pointed out to METH and Me₂SO as those more suitable for cryopreservation. First an exploratory cryopreservation experiment with Me₂SO supplemented with 0.04 M trehalose (TRE) was tested following the protocol for cryopreservation of embryos (8-h blastula) of Bellas and Paredes, 2011, which did not give satisfactory results. A cryopreservation experiment was performed with both cryoprotectants supplemented with 0.04 M trehalose on 4-arm pluteus larvae (48 h-old) developed in these pre-treatment conditions, followed by a simpler and shorter protocol with a cooling rate of 1 °C/min to -35 °C, achieving for the first time the successful cryopreservation of P. lividus larvae. When larvae were incubated in low salinity or low temperature pre-treatments, they showed delayed larval development and abnormalities. In contrast, pretreatments with high temperature and salinity showed good results. Dimethyl sulfoxide with trehalose proved to be the only effective cryoprotectant for successful cryopreservation of P. lividus larvae. The success of dimethyl sulfoxide is consistent with that described for other cases in previous literature, where dimethyl sulfoxide, although not the least toxic compound, gave the best cryopreservation result.