Exploring the feasibility of cryopreserving larvae of the common cockle (Cerastoderma edule) for hatchery production

The decline of natural populations of the common cockle (Cerastoderma edule) through the European coast is posing a threat to local small-scale fisheries. These declines are primarily attributed to the prevalence of several pathogens and the disseminated neoplasia in cockle populations. The institution of a biobank of cryopreserved larvae could enhance hatchery production and help the restocking. The present work aimed at the development of a cryopreservation protocol for larvae of the common cockle using the mollusk cryopreservation protocols designed in our laboratory. Toxicity bioassays and short-term cryopreservation experiments were performed for protocol optimization according with cellular tolerance. Once settled, the viability of cryopreserved larvae was studied long term. Toxicity tests evidenced high tolerance of larvae against detrimental effects of Cryoprotecting Agents (CPAs). Cryopreservation of 48 h-old D-larva showed a 100% survival when increasing the equilibrium time from 15 to 60 min and using Propylene-Glycol (PG) + 0.4 M Trehalose (TRE) in Filtered Sea Water (FSW) and 60 min of exposure to CPA solution before slow-cooling. However, when cryopreserving the older larvae, the variation in equilibrium times hardly showed any effect but 10% Ethylene-Glycol (EG) + 0.4 M TRE and 60 min of exposure yielded the best relative survivorship (100%). Cryopreservation caused a significant delay on the growth rate of the latest larval stage. However, cryopreserved larvae survived to day 4-6, while 30 ± 12.17% of control larvae developed into pediveliger stage, of which 50% settled and transformed into juvenile cockles. These results demonstrated the role of the cell-type specificity in cryopreservation and highlight the importance of studying potential long-term effects of this tool to ensure the viability of the protocols.