Marine bivalves are highly valuable resources, however, some shellfish natural banks are endangered due to factors such as anthropogenic pressure and pathologies. Portuguese oyster (Crassostrea angulata) and striped venus clam (Chamelea gallina) have high socio-economic value and its endangered natural populations require rehabilitation measures. The preservation of these genetic resources through cryopreservation would support aquaculture and natural restocking. Larvae cryopreservation is advantageous over sperm since it allows the availability of diploid organisms immediately after thawing. This work aimed to develop cryopreservation methodologies for C. angulata (n=7-8) and C. gallina (n=5-6) larvae. The effect of two different cryoprotectants (CPAs), namely 20% (v/v) dimethyl sulfoxide (DMSO) and 20% (v/v) ethylene glycol (EG) was assessed prior and after larvae cryopreservation. The composition of all cryoprotectant solutions included 2% (w/v) polyvinylpyrrolidone 40000 MW and 400 mM sucrose in milli-Q water. Each extender was added in a 1:1 (v/v) proportion to D-larvae diluted in artificial seawater (ASW), with a final concentration of 60,000 larvae/ml. Larval morphology, motility and average path velocity (VAP) were evaluated. Samples were cryopreserved after 3 min of equilibration (at 4°C) in 0.5 ml French straws, performed in a programmable biofreezer. Prior to cryopreservation, larval motility and VAP were affected. These results suggest a stress response to the presence of CPAs. In both species the cryopreservation resulted in significantly lower VAP and motile larvae. The cryopreservation of C. angulata reduced significantly the percentage of normal larvae when compared to control. The use of DMSO in both species resulted in significantly higher VAP and post-thaw motile larvae compared to EG. The present work allowed the establishment of the first successful cryopreservation protocol for Portuguese oyster and striped venus clam D-larvae, resulting in viable post-thaw larvae. In future works long term survival of post-thaw larvae should be further investigated to validate its upscaling and applicability for aquaculture and restocking purposes.
Acknowledgements: Supported by 0139_VENUS_5_E (Interreg POCTEP), ASSEMBLE+ JRA2-H2020-INFRAIA-2016-2017 (No 730984), EBB-EAPA_501/2016 (Interreg Atlantic Area) and received national funds through FCT - Foundation for Science and Technology through project CCMAR/Multi/04326/2019. Catarina Anjos and Patricia Diogo were supported by FCT through (SFRH/BD/130910/2017) and (SFRH/BD/97466/2013), respectively.