Development of techniques for cryopreserving giant clam larvae

Giant clams (Tridacna spp.) are keystone reef organisms that play important ecological roles in Indo-Pacific coral reef ecosystems. However, their populations have declined substantially because of overexploitation, habitat degradation, and climate change. Cryopreservation offers a potential strategy for preserving genetic resources and supporting future conservation and restoration efforts. In this study, we evaluated the feasibility of cryopreserving D-stage larvae of Tridacna crocea and T. maxima using a two-step freezing protocol. The effects of different cryoprotectants, concentrations, and equilibration times on post-thaw larval integrity and motility were assessed. Larval development was monitored using microscale culture techniques and microscopic observations. The results demonstrated species-specific responses to cryopreservation treatments, with T. maxima generally exhibiting greater cryotolerance than T. crocea. Ethylene glycol produced the highest larval integrity percentage (LIP) in both species. In addition, glycerol and propylene glycol yielded relatively high LIP values in T. crocea, whereas methanol and dimethyl sulfoxide (DMSO) performed more effectively in T. maxima. Post-thaw motility was observed only in a limited number of treatment groups, with the highest motility recorded in larvae treated with 0.75 M glycerol for 30 min and 0.5 M DMSO for 20 min. In contrast, non-cryopreserved control larvae exhibited a progressive decline in motility during culture, decreasing to 12.1% in T. maxima and 4.0% in T. crocea after 24 h. To our knowledge, this study represents the first investigation of cryopreservation in tridacnid D-stage larvae. Although post-thaw motility remained limited, the successful preservation of larval structural integrity demonstrates the potential of cryopreservation as a conservation tool for giant clams. Further optimization of species-specific cryopreservation protocols will be necessary to improve post-thaw recovery and support long-term genetic resource banking.