Molecular Understanding of Cryopreservation of Marine Invertebrates: Achievements and New Perspectives.

Cryopreservation is a promising biotechnological tool for the long-term conservation of marine invertebrates, yet its molecular impacts remain insufficiently understood. This review synthesizes current findings on how cryogenic procedures encompassing controlled slow-freezing, two-step freezing, and vitrification affect molecular structures in marine invertebrates. Cryoinjury caused by intracellular ice formation and osmotic stress remains the primary challenge, especially in lipid-rich early developmental stages with limited membrane permeability to cryoprotective agents. DNA damage, including fragmentation and methylation pattern alteration, has been reported in mollusks and corals, while variations in lipid phase transition highlight membrane destabilization under cold exposure. Cryopreservation also perturbs mitochondrial activity, leading to adenosine triphosphate depletion and reactive oxygen species accumulation. Protein alterations include degradation, shifts in energy metabolism enzymes, and stress-induced upregulation of heat shock proteins such as HSP70 and HSP90. In parallel, reductions in RNA synthesis and transcriptional responses related to antioxidant and apoptotic regulation have been documented, demonstrating species-specific redox adaptations. Despite these challenges, evidence suggests that surviving cells may recover basal metabolic activity, underscoring the resilience of some taxa. Future studies should integrate molecular biomarkers including DNA integrity, lipidomics, proteomics, and transcriptomics to evaluate cryoinjury mechanisms and optimize species- and stage-specific cryopreservation protocols. Understanding these molecular consequences will enhance the reliability of cryopreservation as an ex situ conservation strategy for marine biodiversity.