Review of molluscan larval cryopreservation and application to germplasm cryobanking and commercial seed production

This review is focused on the status and summary of larval cryopreservation in aquaculture mollusks. A total of 26 publications were identified addressing 14 molluscan bivalve species. Most studies were conducted on trochophores and D-larvae, a few studies were on umbo larvae, and no study was found on pediveligers. Based on the post-thaw viability, there is no general conclusion about the best larval stage for cryopreservation. The research topics of most publications focused on developing or improving cryopreservation protocols through exploring one or more vital factors of the cryopreservation procedure. The most dominant cryoprotectant agents (CPAs) used for molluscan larval cryopreservation were dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG) at final concentrations of 5–15%. Non-permeable CPAs, such as glucose, fructose, sucrose, trehalose, and polyvinylpyrrolidone (PVP), were dominantly used as additives. Most cooling processes started from 0 °C, 4 °C, or larval culture temperature to −10 °C or − 12 °C at −1 °C/ min and holding at this temperature for 5–15 min with or without “seeding”, and then continued to cool to around −35 °C at −0.3 to −2.5 °C/ min (mostly at −0.5 and − 1 °C/ min) followed by a 5–10 min holding period. All frozen samples were then transferred into liquid nitrogen (−196 °C) for long-term storage. Evaluation of post-thaw viability was reported over a wide range of parameters with no standard criteria, for example, motile post-thaw larvae were considered alive but no definition of swimming velocity. A sharp decrease of survival was often reported after a period of culture although immediate post-thaw survivals were reported high in several studies. Overall, no general optimal protocol could be concluded. Post-thaw larvae were reported to survive beyond metamorphosis in Pacific oysters and even to the adult stage and reproduced successfully after 3 years of culture, indicating the promising future of this technology. Till now, no study has been reported on germplasm cryobanking by use of molluscan larval cryopreservation. Pathways and aspects for promoting molluscan larval cryopreservation technology for germplasm repository establishment and commercial seed production were discussed.